HK1186646B - Apparatus and method for non-invasively detecting diseases that affect structural properties in biological tissues - Google Patents

Description

Apparatus and method for non-invasive detection of diseases affecting structural properties of biological tissue

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application relates to and claims the benefit of U.S. provisional patent application nos. 61/410,825, 61/410,827, 61/410,830, 61/410,831, 61/410,833, 61/410,834, 61/410,835, and 61/410,839, all filed on 5/11/2010, and all incorporated herein by reference in their entirety.

Filed concurrently herewith on the same day is a co-pending non-provisional patent application which also claims the benefit of each of the above U.S. provisional patent applications entitled "improved algorithm for detecting diabetes", assigned U.S. patent application number [ to be assigned ], and having at least one co-inventor. The disclosure of the concurrently filed patent application is hereby incorporated by reference in its entirety.

Background

Non-invasive devices and methods for detecting diseases such as diabetes are described. In particular, exemplary embodiments relate to methods and devices suitable for determining the presence, likelihood, progression and/or severity of diabetes in a mammal.

Diabetes mellitus (diabetes mellitus/diabetes) is a type of metabolic disease in which a person suffers from hyperglycemia (polysaccharidoses), either because the body does not produce enough insulin or because the body's cells do not respond to the insulin produced. Diabetes mellitus is a disease arising from a variety of causative factors and is characterized by elevated plasma glucose levels in the fasted state or following administration of glucose in the Oral Glucose Tolerance Test (OGTT). There are two main forms of diabetes: (1) insulin-dependent or type I diabetes mellitus (also known as juvenile onset diabetes, fragile-onset diabetes, insulin-dependent diabetes mellitus (IDDM)) and (2) non-insulin-dependent or type II diabetes mellitus (also known as NIDDM). Type I diabetes most commonly develops in young people, but can occur in adults with the same autoantibodies as type I. Type II diabetes develops most often in middle-aged and elderly people, but may occur in young people. This hyperglycemic condition produces symptoms of polyuria (frequent urination), polydipsia (increased thirst), or polyphagia (increased hunger). Diabetes is an increasingly large problem in developed and developing countries of the world. Since it has now been predicted that about one in every 10 us adults suffers from diabetes, and, according to reports from centers for disease control and prevention, cases of diabetes will proliferate up to 2-fold, even 3-fold, by 2050, up to one in every three people suffering from disease, mainly type II diabetes.

Insulin is a hormone produced by beta cells in the pancreas. The function of insulin is to regulate the amount of glucose (sugar) in the blood, which enters cells through receptors that accept insulin and allow glucose to enter. Once inside the cell, glucose can be used as a fuel. Excess glucose is stored in the liver and muscle in a form known as glycogen. When blood glucose levels are low, the liver releases glycogen, forming glucose. Without insulin, glucose is difficult to enter the cell. In people with diabetes, the pancreas does not produce insulin, or produces too little insulin to control blood glucose, or produces defective insulin. In the absence of insulin, these symptoms progress to dehydration, leading to hypovolemia, increased pulse rate and dry, flushed skin. In addition, ketones accumulate in the blood faster than the body can clear them by urine or exhaled breath. The respiration becomes faster and shallower, and the respiration has a fruity flavor. Other symptoms that indicate progression to ketoacid toxic diabetic coma (DKA) include vomiting, stomachache, and a reduced level of consciousness. People with diabetes are at high risk for debilitating complications such as renal failure, blindness, nerve damage and vascular disease. Although the risk of complications or complication development can be reduced by tight glucose control in combination with drug treatment and lifestyle changes, effective mitigation of complications begins with early detection. The disease leads to serious complications, including hyperglycemia, macroangiopathy, avascular disease, neuropathy, nephropathy and retinopathy. As a result, diabetes adversely affects quality of life. Similarly, uncontrolled type II diabetes results in excessive glucose in the blood, causing hyperglycemia or hyperglycemia.

People with type II diabetes experience fatigue, increased thirst, frequent urination, dryness, itchy skin, blurred vision, slow-healing wounds or pain, more infection than usual foot numbness or tingling. Without treatment, people with type II diabetes will become dehydrated and gradually show dangerously low blood volume. If type II diabetes remains uncontrolled for a long period of time, more severe symptoms can result, including severe hyperglycemia (blood glucose above 600mg) somnolence, confusion, shock, and ultimately "hypertonic hyperglycemia nonketotic coma". Persistent or uncontrolled hyperglycemia is associated with increased and premature morbidity and mortality. Therefore, therapeutic control of glucose homeostasis, lipid metabolism, obesity, and hypertension are critical in clinical management and treatment of diabetes.

Pre-diabetes (i.e., showing no overt clinical signs of diabetes) can exist for 7 or more years before a glycaemic abnormality is detected and after the onset of the disease is shown or diagnosed, as well as early diabetic complications. There is a need for more robust screening of individuals at risk for diabetes. The main reason is that there are no simple, definitive laboratory tests available to identify subjects at risk of developing diabetes or pre-diabetes. There is also a need to identify subjects with diabetic conditions, including pre-diabetic subjects and diabetic subjects, so that they can receive treatment early and non-invasively monitor the progression of the disease over time. Early diagnosis, intensive therapy and consistent long-term follow-up assessments for diabetics are important for effective care, which can help protect vision and significantly reduce the risk of blindness. Diabetes Control and Complications Testing (DCCT) in the united states has shown that complications can be reduced, for example, by eighty percent (80%) if a diabetic can be detected and placed under glucose control. After it becomes apparent that the patient may develop diabetes, the physician is trained to invite the patient back to conduct more tests on a regular basis to determine if the patient's condition has actually developed a disease. The physician has a certain protocol as to how long the patient should wait before being called back to perform more tests. If the patient has few symptoms suggesting diabetes, the patient may not be recalled more than a year. If there are several suggestive symptoms, the physician may wish to recall the patient only a few months later. Unfortunately, there is no diagnostic tool for accurately predicting how long a patient may experience symptoms of diabetes or for determining how much risk a patient actually develops disease. If such a tool is available, the physician can adapt his recall and treatment mode to the needs of the patient.

Modern diabetes screening and monitoring is particularly "punture-intensive" because diabetics have to draw blood to test their glucose levels. The only practical and reliable screening method currently available for monitoring blood glucose is by blood sampling means. The main screening and diagnostic tests currently in use-Fasting Plasma Glucose (FPG) and Oral Glucose Tolerance Test (OGTT) -are not considered optimal because they are inconvenient and unpleasant. Both require venous blood draw and are fasting tests, so they can only be practically implemented in morning appointments and are prone to non-compliance issues. For OGTT, measurements occur two (2) hours after the patient ingests a 75g oral glucose load. Many studies have evaluated the performance of each of these tests in different populations. It is believed that about half of those with diabetes are misclassified by a single FPG test. In addition, it is believed that the OGTT has the disadvantage of relatively poor reproducibility. In addition, the HbA1c test reflects a longer term 90-day glycemia and control or lack of control than FPG, and the test results may also be misinterpreted due to changes in recent diet or hemolytic conditions. Such blood glucose testing methods are of limited value as indicators of long-term glycemic status. In summary, blood glucose measurements (such as HbA1c and FPG) are of limited value as reliable indicators of long-term glycemia status.

Therefore, there is a need for a rapid, accurate, reliable, convenient, and non-invasive screening test as a viable alternative to current tests. Ideally, an improved screening test would measure analytes directly related to disease progression and risk of complications, and the chemical marker as an integrated biomarker would not change for intra-or inter-patient day changes. In addition, the measurement should provide sufficient accuracy to detect early stage diabetes and have sufficient accuracy to obviate the need for repeated, definitive testing. When it becomes apparent that a patient may have diabetes, doctors and optometrists will invite the patient back to conduct more tests on a regular basis to determine if the patient's condition actually develops disease or is identified as diabetic. There is a certain protocol as to how long a patient should wait before being selected for more tests. If the patient has few symptoms suggesting diabetes, the patient may not be recalled more than a year. If there are several suggestive symptoms, the patient may be recalled only a few months later. It would be useful if available diagnostic tools and methods exist for an immediate determination that non-invasively and accurately determine whether a patient is at risk of actually developing diabetes or actually has diabetes.

The main consequence of hyperglycemia is that proteins are hyperglycosylated (non-enzymatic glycation) in a process known as the maillard reaction. Excessive glycosylation ultimately leads to the formation of various protein-protein cross-linked and non-cross-linked structures, known as advanced glycation end products (AGEs). AGEs are believed to present attractive candidate analytes for non-invasive measurement. AGEs have been implicated as a causative factor in complications of diabetes, including Diabetic Retinopathy (DR). Protein glycation is a multi-stage reaction that begins with the formation of sugar adducts of proteins, known as fructosamines or amadori compounds, which mature gradually to form AGEs. Some AGEs require oxidation chemistry for their formation, which is known as sugar oxidation products. Collagen is a protein that readily undergoes glycation and sugar oxidation. Due to its long half-life, the level of AGE in collagen is believed to act as a long-term integrator (integrator) of overall glycemia that is insensitive to fluctuations in short-or mid-term glycemia control. As a result, AGEs naturally accumulate during healthy aging, but at a significantly accelerated rate in people with diabetes. Protein glycation and AGE formation are accompanied by an increase in free radical activity that causes biomolecule damage in diabetes. AGE levels are positively correlated with the severity of retinopathy, nephropathy and neuropathy and, therefore, are indicators of systemic damage to proteins in diabetes and a measure of the patient's risk of diabetic complications. In addition, due to mild to severe hyperglycemia associated with pre-diabetes and type II diabetes, individuals in the early stages of the continuum will accumulate AGEs in their tissues at a higher rate than normal. Thus, given sufficient analytical sensitivity, accurate AGE measurement in an individual offers the prospect of detecting early deviations from normal glycemia. AGE is currently analyzed by invasive procedures that require biopsy samples, and thus, AGE is not used in diabetes screening or diagnosis.

Tissues such as the ocular lens (oculars) can exhibit fluorescence when excited by a light source with an appropriate wavelength. This fluorescence emission, derived from endogenous fluorophores, is an intrinsic property of tissue, known as autofluorescence, to be distinguished from the fluorescence signal obtained by the addition of exogenous markers (such as sodium fluorescein). The tissue fluorophores absorb light of a certain wavelength (excitation light) and release it again (emission) with light of a longer wavelength. Several tissue fluorophores such as collagen, elastin, lipofuscin, NADH, porphyrins, and tryptophan have been identified. Each fluorophore has its characteristic excitation and emission wavelengths, which enables localization and further quantification of the specific fluorophore. Autofluorescence can be induced in several tissues and can therefore be applied in the study of several diseases. It is also used to distinguish malignant from benign tissue in several tissues such as the skin and cervix. In addition, in ophthalmology, autofluorescence of the lens increases with aging and diabetes. Autofluorescence of the lens appears to be caused by glycation and subsequent oxidation of the lens, which forms AGEs. The lens represents a rare biological target because the proteins in the lens are relatively static in life and do not turn over (i.e., undergo retroglycation), allowing for the accumulation of AGEs.

Advances in ocular lens fluorescence spectroscopy have revealed the potential for non-invasive devices and methods to sensitively measure changes in the lens of the eye associated with diabetes. The system relies on illuminating a volume of the lens of a selected human subject (about 1/10 mm) with low power excitation from a monochromatic light source³To about 3mm³Or greater) the emitted fluorescence spectrum. The sensitivity of this technique is based on the measurement of the intensity of the fluorescence in selected regions of the fluorescence spectrum and the normalization of this fluorescence with respect to the attenuation (scattering and absorption) of the incident excitation light. The amplitude of the unshifted rayleigh line, measured as part of the fluorescence spectrum, is used as a measurement of the excitation light attenuation in the lens. It is believed that with this method, normalized lens fluorescence provides for diabetic and non-diabetic patients as compared to more conventional measurements of fluorescence intensity from the lensA more sensitive differentiation between the patient's lenses. The results of such clinical measurements can be used to describe the relationship between normalized lens fluorescence and hemoglobin A1c levels in diabetic patients.

By quantifying light AGEs in the lens or other tissues of the eye, optical spectroscopy offers a potential way to detect diabetes early and non-invasively. In spectroscopy, the instrument emits laser or other light onto the skin or into the eye. Fluorescence spectroscopy (also known as fluorescence or spectrofluorimetry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample by measuring its reflected or emitted light to detect the presence of certain molecules. In fluorescence spectroscopy, a substance (species) is first excited from its ground electronic state into one of various vibrational states in an excited electronic state by absorption of a photon. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state. The molecule then falls back again to one of the various vibrational levels of the ground electronic state, emitting photons in the process. As different molecular species may fall back to the ground state from different vibration levels, the emitted photons will have different energies and thus different frequencies. Those photons that are reflected by the particle surface or refracted through them are referred to as "scattered" photons. Scattered photons may encounter another particle or be scattered off the surface, and they may be detected and measured. Each molecule has a distinct structure that reflects light at a specific wavelength; all glucose molecules share a unique characteristic that is quite different from other blood components such as hemoglobin. If the return wavelength is different from the established standard, the device alerts the patient or physician to the presence of the problematic molecules or cells. Thus, by analyzing the different frequencies of the emitted light in fluorescence spectroscopy and their relative intensities, structures with different vibration levels can be determined.

Fluorescence-based systems rely on the propensity of certain cellular components, called fluorophores (e.g., tryptophan, flavins, collagen), to emit light when excited by light of a particular wavelength, with peak intensities in different but corresponding frequency bands. The actual amount of light emitted by a fluorophore is very small (at the nano-watt level) and requires an extremely sensitive light detection system. The basic function of the optical spectroscopy device is to illuminate the sample with a desired and specific wavelength band and then separate the much weaker emitted fluorescence from the excitation light. Only the emitted light should reach the eye or the detector so that the resulting fluorescent structures overlap with high contrast against a very deep (or black) background. The limit of detection is usually controlled by the background darkness and the excitation light is usually hundreds of thousands to millions of times brighter than the emitted fluorescence.

If AGEs are illuminated by light at 300-500nm, 400-700nm fluorescence is emitted. As AGEs develop, certain early metabolic changes can be detected by fluorescence spectroscopy. Reflectance techniques attempt to characterize tissue by measuring the amount and wavelength of light reflected back to a sensitive light detector when the tissue (e.g., the lens of the eye) is exposed to a light source. Analyzing the fluorescence and reflectance measurements using a computer-based algorithm; however, these systems have not been extensively studied. Noninvasive ocular fluorescence measurements have been investigated in many cases for diabetes screening and AGE quantification.

For example, autofluorescence of the lens of the eye can be measured with a computer fluorescence photometer (Fluorotron Master, Coherent Radiation Inc. (Palo Alto, CA)) equipped with a special lens ("anterior segment adapter") for scanning the lens in detail. Autofluorescence of the lens, excited by a continuous blue beam, can be scanned along the optical axis by moving the inner lens system of the fluorescence photometer by a computer controlled motor. The wavelengths of the excitation light and the fluorescence light may be set by color filters to have transmission peaks at 490nm and 530nm, respectively. The measured autofluorescence, expressed as a fluorescein concentration equivalent, can be recorded as a function of distance in the eye.

It is always desirable to detect disease early in its development. In particular, it is desirable to screen and begin treatment of glucose intolerant individuals as early as possible, even before the onset of diabetes, vascular damage progresses gradually with impaired glucose tolerance. Additionally, β -cell function is severely impaired when significant changes in glucose homeostasis are exhibited, such as Impaired Glucose Tolerance (IGT) and Impaired Fasting Glucose (IFG); therefore, timely intervention is important to maintain the remaining insulin secretory capacity. Early detection enables early treatment, which is generally considered to be of high success, to treat a variety of diseases. More recently, it is believed that analysis of the eye, and in particular the lens of the eye, can produce indications of various types of diseases. For example, measurements of light scattering within the eye have been shown to provide useful diagnostic information to detect and monitor disease progression. Since the thickness of this area is up to a few millimeters, the measurement of this area, in order to be useful, requires very precise information on the measurement location. This is particularly true because the human eye is in almost constant motion even when the patient is gazing at an illuminated target. This is particularly true because eye care professionals, such as optometrists, regularly examine, diagnose, treat, and manage diseases, injuries, and conditions of the eye and related structures, as well as identify related systemic conditions affecting the eye. Optometrists, through their clinical education and experience, and widespread geographic distribution and devices, provide primary eye and vision care to the public. It is often the first time that a health care practitioner examines a patient with undiagnosed diabetes or diabetic eye manifestations.

The efficacy of early intervention in significant disease progression through lifestyle modification or drug therapy is shown by the Diabetes Prevention Program (Diabetes Prevention Program Research Group) NEJM 346:393-403, 2002. However, determination of IGT and IFG is a problem in itself due to the relatively invasive nature of these assessments, particularly the IGT assessment by the Oral Glucose Tolerance Test (OGTT). In addition, another important diagnostic issue is monitoring glucose homeostasis to confirm diabetes. Compliance with glucose monitoring is poor because of the pain and inconvenience of using a lancet for routine blood collection. In addition, non-invasive monitoring techniques for diabetes and determining treatment efficacy are desired. Finally, an assessment of the progression of frangipanis diabetes to complications is only feasible after the complications are well established. Therefore, it would be beneficial to have a method of assessing the progression of diabetes from pre-diabetes and a method of monitoring the progression of the disease.

At least one such attempt is known: a commercial grade non-invasive diabetes detection/screening device was created that measures the lens fluorescence of the eye, referred to as Accu-Chek D-tester. The Accu-Chek-D-tester is essentially a confocal microscope because it uses a confocal optical device to measure AGEs to examine uncontrolled blood glucose levels and early signs of type II diabetes because they accumulate more rapidly in the eyes of individuals with high blood glucose levels than in the eyes of individuals with normal levels. The device applies the so-called biophotonic technique and detects diabetes by emitting blue light to the lens of the patient's eye. The returned light is collected and analyzed. The light emitted from the eyes of people with diabetes is stronger than that of people without diabetes. Specifically, the laser beam passes through a light source aperture and is then focused by an objective lens into a small (ideally diffraction-limited) focal volume within or on the surface of the patient's eye. The scattered and reflected laser light and any fluorescent light from the illumination spot are then reconverged by an objective lens (light collector). The beam splitter, which separates part of the light into the detection device, can have such a filter in a fluorescence confocal microscope: the filter selectively passes the fluorescence wavelengths while blocking the original excitation wavelengths. After optionally passing through a pinhole, the light intensity is detected by a light detection device, such as a photomultiplier tube (PMT), which converts the light signal into an electronic signal that is recorded by a computer for further analysis. Specifically, the Accu-Chek D-tester emits blue light to the lens of the eye, and then condenses and analyzes the returned light.

However, the main disadvantages of the Accu-Chek-D-Tecter are its relative slowness, inaccuracy and high manufacturing cost. While the device is said to obtain readings (fluorescence, 15 seconds; backscatter, 15 seconds) within 30 seconds to obtain a ratio of fluorescence signal to backscatter signal from a particular location within the patient's lens, the device employs a sliding filter converter to select either green (fluorescent) or blue (backscattered) light that strikes a photodetector through a crank mechanism. Rotation of the stepper motor causes a two-position slider (slider) to take one or more seconds to move from one filter to the other. In addition, in use, the patient is required to self-align with the device via the gaze system, which makes it difficult and time consuming.

Most non-invasive analyzers are not specifically designed for high throughput screening purposes. They are difficult and expensive to bind to high throughput screening environments. Even after the analyzer is incorporated into a high throughput screening environment, there are often many problems, including increased probability of system failure, data loss, time delays, and loss of high cost compounds and reagents. Thus, existing non-invasive diabetes detection devices generally have not recognized the need to provide analytical flexibility and high performance.

Typically, non-invasive devices utilize some form of spectroscopy to obtain a signal or spectrum from the body. Spectroscopic techniques include, but are not limited to, Raman and Rayleigh fluorescence, as well as techniques that utilize light from ultraviolet to infrared [ ultraviolet (200 to 400nm), visible (400 to 700nm), near infrared (700 to 2500nm or 14,286 to 4000cm-1), and infrared (2500 to 14,285nm or 4000 to 700cm-1) ]. It is important to note that these techniques differ from the traditional invasive and alternative invasive techniques listed above in that the sample analyzed is part of the human body in situ, rather than a biological sample obtained from the human body.

There is a real need for a versatile, sensitive, high throughput screening apparatus and method that can handle a wide variety of assays and a wide range of patients while reliably maintaining high sensitivity levels. In addition to early identification, there is a need for a diabetes detection device, apparatus, method and/or system for detecting diabetes that does not require fasting and is not exposed to cumulative tests of glucose level changes caused by various causes, including food, stress-specific drugs or short-term changes in diet and exercise.

Description of the exemplary embodiments

Various exemplary embodiments of the invention will now be described with or without reference to the accompanying figures, in which like reference numbers indicate identical or functionally similar elements. Example embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations. Thus, the following more detailed description of the exemplary embodiments, as described and/or illustrated in the figures, is not intended to limit the scope of the claimed subject matter, but is merely representative of exemplary/example embodiments.

Certain aspects, advantages, and novel features are shown in the drawings and/or described herein. It should be understood that all such aspects, advantages, and features explicitly or inherently discussed herein are not necessarily applied or implemented in accordance with any particular embodiment or aspect thereof. Thus, for example, those skilled in the art will recognize that the exemplary embodiments may be implemented in a manner that achieves one advantage or group of advantages as taught or inferred herein without necessarily achieving other advantages as may be taught or suggested herein. Of course, advantages not expressly taught or suggested herein may also be realized in one or more exemplary embodiments.

The following rules of interpretation apply to this description (written description, claims and drawings) unless otherwise indicated: (a) all words used herein are to be interpreted as having such a part of speech or number (singular or plural) as is required for each case; (b) as used in the specification and the appended claims, the singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise; (c) the antecedent "about" applied to such ranges or values indicates an approximation within the deviation of the range or value known or expected in the art by measurement methods; (d) the terms "herein," "its," "up to this point," "above," and "below," and words of similar import, refer to this specification as a whole and not to any particular paragraphs, claims, or other sections unless otherwise specified; (c) descriptive headings are for convenience only and thus do not control or affect the meaning or structure of any portion of the specification; and (d) "or" and "any" are non-exclusive, while "including" is non-limiting. Furthermore, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Where a specific range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within that range. All smaller subranges are also included. The upper and lower limits of these smaller ranges are also included in the range, subject to any specific exclusion limit within the stated range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can also be used, the preferred methods and materials are now described.

As used herein, the terms "an embodiment," embodiments, "" the embodiment, "" the embodiments, "" one or more embodiments, "" some embodiments, "" certain embodiments, "" one embodiment, "" another embodiment, "and the like mean" one or more (but not necessarily all) embodiments of the disclosed apparatus and/or method, unless expressly specified otherwise.

The term "determining" (and grammatical variants thereof) is used in an extremely broad sense. The term "determining" includes various operations, and thus "determining" can include calculating, computing, processing, inferring, researching, viewing (e.g., viewing a table, a database, or another data structure), ascertaining, and the like. Likewise, "determining" can include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), and the like. Likewise, "determining" may include resolving, selecting, establishing, and the like.

The phrase "based on" does not mean "based only on," unless explicitly stated otherwise. In other words, the phrase "based on" describes both "based only on" and "based at least on".

The word "exemplary" or "example" is used exclusively herein to mean "serving as an example, instance, or illustration. Any embodiment described herein as "exemplary" or "exemplary" is not necessarily to be construed as a preferred embodiment or as advantageous over other embodiments.

As used herein, the terms "user" or "patient" or "subject" are used interchangeably, and the above terms include, but are not limited to, humans, whether or not under the care of a physician, and other mammals. As used herein, the terms "eye scan", "scanning an eye", "scanning multiple eyes" are widely interchangeable terms that generally refer to the measurement of any, substantially all, or all portions of an eye, including but not limited to the lens of the eye or any other tissue or nerve associated with the eye.

The embedded computer subsystem may include at least one Central Processing Unit (CPU) or "processor", memory, storage, a display, and a communication link. An example of a CPU is an Intel Pentium microprocessor. The memory may be, for example, static Random Access Memory (RAM) and/or dynamic random access memory. The storage device may be implemented using non-volatile RAM or a disk drive. Liquid crystal displays are an example of the type of display that can be used in the device. The communication link may be a high-speed serial link, an ethernet link, or a wireless ("WiFi" or "broadband") communication link. The embedded computer subsystem may generate, for example, disease status forecasts from the collected data, perform calibration maintenance, perform calibration transfers, run instrument diagnostics, store past analysis history and other pertinent information, and in some embodiments, may communicate with a remote host to send and receive data and new software updates. The communication link may be used for medical billing based on the number of tests performed on each device. It can also be used for customer service to track failure or error rates on each device.

The embedded computer system may also contain a communication link that allows the prediction record and corresponding spectrum of the object to be transferred to an external database. In addition, the communication link may be used to download new software to the embedded computer, update the multivariate calibration model, provide information to the subject to enhance management of their disease, etc. Embedded computer systems are very similar to information applicators. Examples of information applicators include personal digital assistants, web-enabled cell phones, and laptop computers. The communication link may be used for medical billing based on the number of tests performed on each device. It can also be used for customer service to track failure or error rates on each device.

In further example embodiments, the biomicroscope apparatus may be configured with, connected to, or in communication with a system for automated remote monitoring of the operational status of one or more biomicroscopes disclosed herein, each having a computer therein for determining device status information (e.g., usage counts, usage billing/accounting for exceeding contact minimum (contact minimum), hardware or software error coding, storage or database operations for remote system diagnosis of points of failure, capturing service response time until performance recovery, etc.), including interventions in the biomicroscope to intercept status information from the computer and pass it to an interface for capturing and communicating status information to a remote location, a communication link between the interface for capturing and communicating information and the remote location, a computer, And a computer at a remote location to process the information. The system interrogates a biological microscope with a scanner. A scanner cooperating with the central computer may interrogate and monitor each biomicroscope at a uniform rate, or upon request by a user at the central location, alter the interrogation rate at which one or more biomicroscopes interrogate selected regularly-increased biomicroscopes, slowing the interrogation rates of other biomicroscopes, to provide real-time monitoring of the selected biomicroscopes. Based on the results of the scanning or interrogation procedure, the system may be configured to provide voice and speech capabilities to provide the operator with the option to communicate "live" with a customer service representative of the vendor or manufacturer of the biological microscope for troubleshooting issues. The system is configured to utilize centralized computing and routing and or "cloud" computing or storage.

"software" and "machine-readable code operable on an electronic computer" are synonymous and refer to software or hardwired instructions for controlling a logical operation of the computer. The term computer or processor refers to an electronic computer or its specific logic processing hardware. The machine-readable code is embodied in tangible media, such as hard disks or hardwired instructions.

The processor in the system may be a conventional microcomputer having a keyboard and mouse input device, a monitor screen output device, and a computer interface operatively connecting the system components, including, for example, an eye tracking component or device, robotic elements, and the like.

It should also be understood that all measurements are approximate, which is provided for the description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and/or examples are illustrative only and not intended to be limiting.

Some features of the embodiments disclosed herein may be implemented as computer software, electronic hardware, or combinations thereof. To illustrate this interchangeability of hardware and software, various components may be described generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system, as can be readily obtained by a skilled artisan. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the claims.

Where the described functionality is implemented as computer software, such software may include any type of computer instructions or computer-executable code or algorithms that are located or stored in (even transitory) memory devices and/or transmitted as electronic signals over a system bus or network. Software that implements the functionality associated with components described herein may comprise a single instruction, or many instructions, and may be distributed over several different code segments, among different programs, and across several memory devices.

As used herein, "determining a disease state" includes determining the presence or likelihood of diabetes; the degree of diabetes progression; changes in the presence, likelihood, or progression of diabetes; probability of having, or not having, developing, or not developing diabetes; the presence, absence, progression or likelihood of diabetic complications.

"Diabetes" includes some blood glucose-regulating conditions including type I, type II and gestational Diabetes, other types of Diabetes recognized by the american Diabetes association (see ADA Committee Report, Diabetes Care,2003) and similar regulatory agencies, hyperglycemia, impaired fasting glucose, impaired glucose tolerance and pre-Diabetes. The ocular tissue reflectance characteristics include any reflective property of tissue that is useful in the correction of detected light that has been found to be useful for assessing intrinsic fluorescence and rayleigh scattering spectra of tissue.

"measurement of chemical changes due to glycemic control" means any change in histochemical characteristics due to glycemic control, examples including concentration, measurement of the presence of glycated end products in ocular tissues, concentration, or change in concentration; a measure of the rate of accumulation or change in the rate of accumulation of such end product;

"measurement of glycation end-products" means any measurement of the presence, time, extent or status of ocular tissue associated with hyperglycemia, including as an example the measurement of the presence, concentration or change in concentration of glycation end-products in the tissue; a measure of the rate of accumulation or change in the rate of accumulation of such end product; measurement of the presence, intensity or change in intensity of fluorescence intensity and rayleigh backscattering, alone or in combination, known to be associated with tissue glycation endproducts; and a measure of the rate of accumulation or change in the rate of accumulation of such signals. When light is described as having "a single wavelength," it should be understood that light may actually include light at multiple wavelengths, but a significant portion of the energy in the light is transmitted at or near the single wavelength.

For example, there are some non-invasive methods for analyte concentration determination. These methods are very different, but each have at least two steps in common. First, a reading is obtained from the body using the device without obtaining a biological sample. Second, the algorithm converts the reading into an analyte (e.g., glucose) concentration estimate. One example of a non-invasive analyte concentration analyzer includes those based on spectral concentration and analysis. Typically, non-invasive devices utilize some form of spectroscopy to obtain a signal or spectrum from the body. Spectroscopic techniques include, but are not limited to, Raman and fluorescence, as well as techniques that utilize light from ultraviolet to infrared [ ultraviolet (200 to 400nm), visible (400 to 700nm), near infrared (700 to 2500nm or 14,286 to 4000cm-1), and infrared (2500 to 14,285nm or 4000 to 700cm-1) ]. A specific range for non-invasive analyte determination in diffuse reflectance mode is about 1100 to 2500nm or a range therein. It is important to note that these techniques differ from the traditional invasive and alternative invasive techniques listed above in that the sample analyzed is part of the human body in situ, rather than a biological sample obtained from the human body.

Three modes are commonly used to collect non-invasive scans: transmission, transmission reflection (transmission) and/or diffuse reflection. For example, the concentrated light, spectrum, or signal is light transmitted, diffusely reflected, or transreflected (transflected) through a body region. Transflective refers to the collection of signals not at the point or area of incidence (diffuse reflection), nor on the opposite side of the sample (transmission), but at some point or body area between the transmissive and diffusely reflective convergence regions. For example, the transmitted reflected light enters a fingertip or forearm in one area and exits the other area. When sampling skin tissue with near infrared, the transmitted and reflected radiation is typically dispersed radially from the incident photons by 0.2 to 5mm or more, depending on the wavelength used. For example, light that is strongly absorbed by the body, such as light near the maximum water absorption at 1450 or 1950nm, is concentrated after a small radial divergence for detection, and less absorbed light, such as light near the minimum water absorption at 1300, 1600, or 2250nm, is optionally concentrated at a larger radial or transmitted reflection distance from the incident photons.

The example embodiments described herein constitute improvements to one or more of the methods and apparatus disclosed in the following patents (purportedly describing the above Accu-Chek D-controller design), the entire disclosures of which (written description and drawings) are incorporated herein by reference.

U.S. Pat. No. 5,203,328 to Samuels entitled "Apparatus And method For Quantitatively Measuring Molecular changes in an Ocular Lens" (Apparatus And Methods For quantitative Measuring Molecular changes in The Ocular Lens) ". This patent discloses an apparatus and method for determining whether a patient has diabetes. The system and method measure a characteristic of the patient's eye indicative of diabetes. Specifically, the system and method irradiate ocular tissue of a patient's eye and measure backscattered light and fluorescent radiation produced by the ocular tissue in response to the excitation light. The intensity of the backscattered light and the fluorescence light of the specific wavelength is then used to determine whether the patient suffers from diabetes.

U.S. Pat. No. 5,582,168 entitled "Apparatus And method For Measuring Characteristics Of Biological tissues And Similar Materials" (Apparatus And Methods For Measuring Characteristics Of Biological tissue And Similar Materials) ". This patent illustrates such an apparatus and method: which combines two or more measurement techniques to achieve a more accurate final assay by measuring biological tissue and similar material characteristics. These are described in relation to apparatus and methods for measuring the eyes of a person. In addition, the correction method described therein comprises measuring elastically scattered excitation light. Samuels describes a simple linear correction technique.

U.S. Pat. No. 6,088,606 entitled "method and apparatus for determining the duration of a medical condition". This patent discloses systems and methods for determining the duration of a medical condition and methods relating to determining the duration of a disease rather than for diagnosing or screening for the presence of a disease or for quantifying the concentration of a specified chemical analyte.

U.S. Pat. No. 4,895,159, entitled "Diabetes Detection methods" and U.S. Pat. No. 4,883,351, entitled "Apparatus for detecting Diabetes and Other Abnormalities affecting the Lens of the eye" (Apparatus for the Detection of Diabetes and Other Abnormalities affecting the Lens of the eye) "each discloses systems and methods for detecting the presence of Diabetes using only backscattered light.

Fig. 1 shows a side view of an eye 10. Eye 10 includes cornea 11, iris 12, pupil 14, lens 15, retina 16, and optic nerve 17. Light enters the eye through the pupil 14, is focused, inverted through the cornea 11 and lens 15, and projected onto the retina 16 at the back of the eye. The iris 12 acts as a shutter (shutter) that can be opened or closed to regulate the amount of light entering the eye through the pupil 14.

Eye 10 is composed of four tetrad (quadratants) sections associated with the optic nerve head: (a) a temporal portion consisting of quadrants facing the temporal portion of the skull, (b) an upper portion consisting of quadrants above the optic nerve head, (c) a nasal portion consisting of quadrants facing the nose, and (d) a lower portion consisting of quadrants below the optic nerve head. In one aspect, measurements of a particular quadrant or quadrants of the ocular lens, i.e., temporal, superior, nasal, and/or inferior quadrants, can be collected/used to generate data regarding structural features of the eye. In other words, in an example method of optically detecting AGEs in the lens of a subject's eye, the subject's eye may be exposed to a fixation point. Exposing the subject's eye to the excitation light source may include directing light to a desired portion of the subject's eye. Directing light to a desired portion of the subject's eye may include directing light to a nasal portion, a temporal portion, an upper portion, or a lower portion of the lens. It may also include directing light to other parts or tissues of the eye such as, but not limited to, the retina, vitreous, crown (corona), etc.

In an example embodiment, an ocular lens fluorescence biomicroscope is provided for use by ophthalmologists, optometrists and other health care professionals trained in routine eye examination, configured to aid in the diagnosis of diseases affecting properties of the lens structure. The instrument includes an optoelectronic unit and a computerized system for data acquisition and processing.

Fig. 2 through 5A depict perspective, top, and side views, respectively, of an example embodiment for an example biomicroscope with optics comprising a blue LED illumination source, confocal illumination, and collector optics, with the ability to scan a measurement volume through a lens, dichroic filter, and detector that simultaneously measures lens autofluorescence and scattered light from an area region. In addition, there is a red flashing (red blanking) LED target fixation light, arranged within a red flashing concentric ring to help the patient self-align; three IR LED lights to illuminate the eye; and a camera. Specifically, the components of the optical device unit include:

1. biological microscope light source

a. Blue (e.g. 465nm) LED excitation light

b. Hole(s)

c. Band-pass filter (430-470 nm)

2. Biological microscope focusing light device

a. Source lens

b. Collector mirror with IR light-blocking filters

3. Biological microscope light detector

a. A silicon photomultiplier has a preamplifier, a Peltier cooler, and a power supply.

b. A front surface mirror.

c. A stepper motor driven filter wheel with 25% medium density, and long pass (long pass) (500-1650nm) filters.

4. Positioning light device

a. Red flashing LED-fixation light, visible within the red flashing concentric ring, is provided by the cavity of the LED-holding tube (highlighted by the deeper line in fig. 3 and 4).

b. Three IR LED lights for camera illumination

c.IR sensitive CCD camera for locating pupil

5. A fluorescent reference target that can be placed in the optical path during a self-test procedure at start-up.

In an example embodiment, an automated tracking procedure is provided for locating the pupil of a patient's eye. An operator (e.g., a health care professional or assistant) positions the subject's eyes so that they are focused on the computer screen, and the system automatically aligns its optical axis before taking the measurement. The operator knows that the eye is being tracked by the pupil tracking system because the radial lines appear on the screen within the circle around the pupil and a smaller circle appears within the pupil. The patient is instructed to close and open the eye (to wet the cornea with the tear film) to reduce blinking, and the operator clicks the start icon to begin the scan. The blue LED light sources are focused to achieve a focused excitation beam of blue light that is initially disposed behind the posterior lens capsule. The light collection optics were aligned confocal within a 1mm diameter and 3mm long measurement volume through which the scan was performed. The following figures describe example embodiments that show or identify various aspects and features disclosed herein.

In the exemplary embodiment, the primary functional components are the light device unit and the laptop personal computer, which run any suitable operating system. The only components that contact the patient are shown in the following perspective views, namely a manually adjustable headrest (in/out) and a motorized adjustable chin rest (up/down). The motorized adjustable optical window (right/left movement) does not contact the patient.

In operation, the example embodiments are configured to project a focused blue light beam onto a patient's lens and non-invasively measure autofluorescent green light from the lens. To accommodate the measurement of the effect of blue light absorbed by the lens, the example fluorescence biomicroscope was configured to measure the scattered blue light and calculate the ratio of autofluorescence to scattered light ("fluorescence ratio"). The clinician may compare the patient's fluorescence ratio to a range of fluorescence ratios expected for the patient's age. By identifying patients with significantly higher fluorescence ratios than expected, clinicians can identify signs of degenerative structural changes in the patient's lens, identify potential risks of chronic systemic disease in combination with other data collected in routine eye examinations, and establish an appropriate patient management plan.

In exemplary systems and methods, a lens of an eye is illuminated with excitation light, and fluorescent emissions of lens tissue in response to the excitation light are detected. Different characteristics of the fluorescence emission, including fluorescence emission intensity or fluorescence lifetime, can be determined. The measured characteristic of the detected fluorescent emission is then compared to an expected characteristic of the fluorescent emission. Detecting the amount of deviation of the fluorescence signature from the expected fluorescence signature is used to determine the duration of time that the patient has experienced the medical condition. In some examples, the backscattered portion of the excitation light may also be used for determination. Measuring AGE strength in the lens of the subject's eye can provide further benefits. For example, if multiple measurements are taken over time, these measurements may be used to monitor the subject's reduced response to dietary intervention strategies, nutritional supplements, medications, external oxidative stress factors such as smoking, and/or other factors. Additionally, measuring AGE intensity or subject lens severity can provide a research tool for studying the correlation between AGE and disease in a large population of subjects.

In an example embodiment, the forehead of the patient is centered on a headrest, which is adjustable by a headrest knob. The patient's eye was illuminated with three near infrared 880nm LED lights and observed with an IR sensitive CCD camera. The eye images are displayed on a computer screen to assist the operator in aligning the patient. The headrest is configured to be manually adjusted to bring the corneal plane of the patient's eye into proximity with the optical window so that the eye focuses on the computer screen in the camera image. Patient self-alignment is indicated by having a red flashing LED looking to center the light around a concentric ring of red flashes. Using a computer interface, the operator can adjust the jaw vertical and horizontal positions of the optical window to enable the patient to sit comfortably while the fixation target is properly positioned. By clicking on the arrow icon on the computer screen, which controls the stepper motor, the operator adjusts the optical window and the jaw rest.

In a further exemplary embodiment, the optical path is aligned with the subject's eye by moving the jaw rest, headrest, and optical axis until the target is centered in the pupil and the iris is in focus. The appropriate focus is determined by an IR camera viewed by the operator (health care worker). The patient fixates on the target to ensure stability of the eye and to relax accommodation. The target remains visible during the scan.

The computer is configured to include software to control an automatic tracking program for locating the pupil. The operator positions the patient's eye so that it is focused on the screen, and the system automatically aligns its optical axis before taking the measurement. As the patient's eye is tracked, the radial lines appear within an alignment circle on the computer screen around the pupil, and a smaller circle appears within the pupil. The patient is then instructed to close and open the eye (to wet the cornea with the tear film) to blink, and the operator clicks the start icon to begin the scan. The blue LED light source is focused to achieve an excitation beam of converging blue light, which is initially disposed just behind the posterior lens capsule. The light collection optics were arranged confocal within a 1mm diameter and 3mm long measurement volume that was scanned through the lens in 0.31mm steps (step). In the eye, blue light is scattered by elastic (rayleigh scattering) and inelastic (fluorescent) interactions with lens proteins such as AGEs.

In the detection path, the filter suppresses the red and infrared light from localizing the infrared LED. The rotating filter wheel optionally chops the beam into blue and green (primarily rayleigh scattered) and green (fluorescent) segments. The alternating scattered and fluorescent light is focused on a high sensitivity silicon photomultiplier and the signal is sent to an a/D converter on the control board of the optical device and then to a computer.

Under software, the measurement volume at the focus of the light source and detector is scanned just behind the posterior lens capsule, through the lens, through the anterior lens capsule, to the aqueous humor, and back again. Computer software records the scattered and fluorescent light during the forward and reverse scans and constructs a graph of each, which is displayed on a computer monitor. The software examined the anterior and posterior surfaces of the lens capsule on the chart, evaluated the apparent thickness of the lens, and averaged the ratio of the autofluorescence of the lens to the scattered light from the central portion of the lens. The software checks that the apparent lens thickness is within the physiological range. The software also detects anomalies in the scans, such as depreciation of the eyes, which can cause inaccurate measurements or excessive differences between scans. For a valid scan, the fluorescence ratio is reported; otherwise, for irregular scans, an error code is reported on the computer monitor and no fluorescence ratio is reported. In the event of an error code, the clinician may then rescan the eye. If the scan is not valid, the software program generates a report that: the report is displayed on a screen and may be printed for the patient and/or the patient folder. The scan data is also automatically saved on the computer hard disk.

In this example embodiment, there is an advantageous gaze target system configuration that overcomes another disadvantage of the Accu-Chek D-tester, namely, in use, seating the patient and requiring the patient to place their forehead on a fixed headrest provided and gaze a visible gaze target located therein. In the case where patient/system interactions are of interest, any intentional or unintentional movement of the patient's eye during treatment may significantly alter the alignment of the eye, relative to the accuracy of the detection. It is therefore necessary for the patient to have their eye fixed during the test. The purpose of the gaze target system is to ensure that the patient looks within narrow limits along the desired line of sight and that the position of the eye when viewed by the instrument camera is well defined. This is done by presenting a visual fixation target to the patient so that the patient's eyes rotate at the desired angle-superior and nasal. The visible fixation target encourages the patient to fixate on the target. The optical axis of the patient's line of sight is displaced upward by about 15 degrees and inward by about 15 degrees to avoid specular reflections from the eye from affecting the fluorescence measurement.

First, the target may not be visible to the patient because the optical axis is not sufficiently aligned with the eye. The operator, while viewing the patient's eyes displayed on the computer screen, adjusts the button controls on the computer screen to bring the viewing and illuminating light means to the central region of the eyes. In Accu-Chek DTectror, the patient gazes at a 0.5mm diameter red LED target located at a distance of about 150mm, which is viewed through a 4mm diameter aperture. Due to the narrow angle accuracy required to see even the LED target, it is often difficult for the patient to locate the hole and LED target because the patient is essentially required to look at the LED target through the rod (straw) when he cannot even see the proximal end of the rod, and there is no visual cue as to where to move his head and where to see the alignment achieved. The anterior and posterior capsule boundaries are automatically noticed as the eye is scanned along the visual axis of the lens. After positioning the anterior and posterior lens locations, the system then scans along the visual axis of the lens setpoint from which fluorescence data can be recorded.

In another example embodiment, which may provide more accurate measurements, the fluorescence intensity is normalized using backscattered excitation light to account for various factors. Such factors may include changes in opacity of the target tissue, which may vary with age or condition of the patient. In such a system, the detector would be able to determine the intensity of fluorescence generated by the target tissue as well as the intensity of excitation light backscattered from the patient's eye. Since the fluorescence will typically have a different wavelength than the backscattered excitation radiation, the detector may be configured to detect the intensity of light returning from the target tissue at the respective different wavelengths of the excitation light and the fluorescence. The ratio of the fluorescence intensity to the intensity of the backscattered excitation light is then determined, thereby normalizing the peak intensity of the fluorescent component. The normalized fluorescence intensity is then compared to an expected normalized fluorescence intensity to determine the duration of time that the patient has experienced the medical condition.

Changes in opacity or transmissivity of the target tissue (e.g., the lens of a patient's eye) can affect the amount of excitation light actually delivered to the target tissue and the amount of fluorescence escaping the target tissue and detected by the detector. Normalizing the fluorescence by the backscattered light produces a measurement of fluorescence that automatically accounts for variations in the energy of the excitation light actually delivered to the target tissue and variations in the amount of fluorescence that escapes the patient's eye after the fluorescence is produced. In a specific example embodiment, where the intensity of fluorescence returned from the target tissue is normalized, the rayleigh component of the backscattered excitation light is used for normalization.

Alternatively, a simpler embodiment can be created that is configured to measure only the fluorescent components and not the backscattered signal, thereby eliminating the need for a filter wheel. For this configuration, fluctuations in LED intensity over time may require a reference detector or calibration target within the device. Fluorescence can be measured at a specific time/delay after the excitation pulse (typically 1-10ns), which eliminates the need for a bandpass filter, only the delay of the detector measurement. In addition, a dichroic beamsplitter arrangement can be used in place of the filter wheel to separate the fluorescence and scatter signals for measurement on separate detectors, thereby saving space.

Apertures may be used on the excitation and collection optics to control the size of the sample volume produced in the human lens. The sample volume should be maximized to increase the photon count and measured SNR, but should be no greater than the human lens (3-5mm thick).

The need for motion control for tracking the pupil can be obviated by a handheld configuration that is secured to the subject by use of a suitable device, such as a sight glass or forehead rest. In use, the operator manually positions the video object over the pupil. The optical element should be configured to scan continuously or individually through the human lens using a mechanical oscillator (e.g., voice coil, piezo, motion progression) and provide the ability to analyze each scan and tell the operator when a successful scan is obtained. The excitation, collection and video axes can be combined to share a common lens, which can be more easily scanned.

In further embodiments, the LED array and detector pair may be configured to form an array of sample bodies, such that no mechanical motion is required for scanning the sample bodies. The optimal sample volume may thus be selected from a given LED/detector pair.

Furthermore, it would be desirable to have a method or apparatus based on portable, handheld, powerful, cost effective, non-invasive and fast imaging configured for detecting fluorescence signals by rayleigh or raman scattering from a "measurement volume" where, for example, confocal beams intersect for objective assessment of eye tissue. Such methods or devices can detect changes in biological, biochemical and cellular levels to quickly, sensitively and non-invasively detect or diagnose the presence of a pre-diabetic condition at an early stage. Such portable methods, devices or apparatus described herein have commercial potential.

After determining which wavelength to use for the light source and examining which part of the recorded spectrum to measure the fluorescence response, it is possible to design a simpler, dedicated system, which is capable of making the same measurements. This can be accomplished by using custom light devices to simultaneously pass and converge light, with a directed optical path to a separate light detector being achieved by appropriate configurations of, for example, filters and dichroic beam splitters. The optical efficiency can be increased by orders of magnitude by comparison with a spectrometer and diode array connected to a filter.

In further exemplary embodiments, the portable (handheld) device may generally include the following features: (i) one or more excitation/illumination light sources and (ii) a detector device (e.g., a digital imaging detector device, or (fluorescence signal detected by Rayleigh or Raman scattering from a "measurement volume" where the confocal beam intersects) may be combined with one or more optical emission filters or spectral filtering mechanisms, and may have a viewing/control screen (e.g., a touch sensitive screen), an image capture and magnification control device The LED array emits light at a wavelength (as described above), such as, but not limited to, about 430 to about 470nm, and the excitation/illumination light source may be coupled with an additional bandpass filter to remove/minimize side spectral bands of light output from the LED array so as not to cause light leakage into the imaging detector with its own filter. The digital imaging detector device may be a digital camera, for example having at least IS0800 sensitivity, but more preferably IS03200 sensitivity, and may be combined with one or more optical emission filters or other equivalent (e.g. miniaturized) mechanized spectral filtering mechanisms (e.g. acousto-optic tunable filters or liquid crystal tunable filters). The digital imaging detector device may have a touch sensitive viewing and/or control screen, an image capture and magnification control device. The case may be an outer hard plastic or polymer shell that encloses the digital imaging detector device with buttons so that the necessary device controls can be easily accessed and manipulated by the user. A micro heat sink or small mechanical fan or other heat sink may be embedded into the device to allow excess heat to be removed from the excitation light source, if desired. The complete device, including all its accessories and accessories, may be powered using standard AC/DC power and/or by a rechargeable battery pack. The complete device may also be connected or mounted to an external mechanical device (e.g., a tripod or a movable stand with a swing arm), allowing mobility of the device in the clinical room without manual manipulation of the device. Alternatively, the device may be fitted with a mobile stand so that it is portable. The device may be cleaned using a wet gauze moistened with water, while the handle may be cleaned using a wet gauze moistened with alcohol, consisting of any suitable antimicrobial hard plastic. The device may include software that allows the user to control the device, including controlling imaging parameters, image visualization and storage of fluorescence and rayleigh scattering as one objective value, image data or measurement values and user information, transmission of images and/or related data, and/or associated image analysis (e.g., diagnostic algorithms) and detection of fluorescence signals by rayleigh or raman scattering from a "measurement volume" where the confocal beams intersect.

As detection efficiency increases, the source intensity can be reduced accordingly by applying low power, short arc lamps, while adequate optical power can be provided by appropriate optical means and filters. Other suitable sources include laser diodes connected to frequency doubling devices, blue LEDs, and filtered, special purpose incandescent lamps. To exclude specular reflections from the detector, polarizing filters are proposed for the transmitting and receiving light means. In addition, the electronics associated with the detection and processing include two similar preamplifiers and a single chip microcontroller, which are used with the detector, equipped with a digital (ND) converted on-board analog. The embedded firmware may direct the operator through the measurement event and then display the processed measurement information on the system's own digital display, or record the data to a computer through, for example, a serial interface.

Devices and methods for fluorescence-based monitoring are disclosed, in some aspects, the devices include optical (e.g., fluorescent and/or reflective) devices for real-time, non-invasive imaging of biochemical and/or organic substances. The device may be small, portable and/or hand-held and may provide high resolution and/or high contrast images. The imaging device can quickly and conveniently provide important biological information to the eye region of a clinician/health care worker. The device may also facilitate image-guided swab/biopsy sample acquisition, targeted and activated optical (e.g., absorption, scattering, fluorescence, reflectance) imaging of exogenous molecular biomarkers. Fluorescently labeled therapeutic agents can also be detected to measure drug interactions and therapeutic compliance, allow for in vivo or in vitro agent comparison, and may allow for longitudinal monitoring of therapeutic response to adaptive interventions in diabetes management. By developing wireless capabilities with dedicated image analysis and diagnostic algorithms, the device can be seamlessly integrated into a remote medical (e.g., electronic health) infrastructure for remote access to health care professionals. Such devices may also have applications beyond diabetes or eye care, including early detection of cancer, monitoring of emerging photodynamic therapy, detection and monitoring of stem cells, and as an instrument in dermatology and cosmetic clinics, among other applications.

In some aspects, there is provided an apparatus comprising: for detecting fluorescent signals based on fluorescence imaging and using rayleigh or raman scattering from a "measurement volume", where the confocal beams intersect, and for monitoring a target, comprising: a light source emitting light for illuminating a target, the emitting light comprising at least one wavelength or wavelength band that causes at least one biomarker associated with the target to fluoresce; and a photodetector for detecting fluorescence. In other aspects, kits are provided for fluorescence-based imaging and monitoring of a target, comprising: the above-described device; and a fluorescent contrast agent for marking the biomarkers at the target, the fluorescence wavelength or band of which is detectable by the device. In still other aspects, methods are provided for fluorescence-based imaging and monitoring of a target, comprising: illuminating the target with light emitted by a light source, the light emitted by the light source having at least one wavelength or wavelength band that causes at least one biomarker to fluoresce; and detecting fluorescence of the at least one biomarker with an image detector.

One example embodiment of an apparatus is a portable optical digital imaging device. The device may utilize a combination of white light, ocular tissue fluorescence and reflectance imaging, and may provide real-time assessment, recording/certification, monitoring and/or care management. The device may be hand-held, small and/or lightweight. The apparatus and method may be adapted to monitor eye tissue of humans and animals. Without limitation, the device may include a power source such as an AC/DC power source, a small battery pack, or a rechargeable battery pack. Alternatively, the device may be adapted to be connected to an external power source. The device may be hardened or contain suitable shock absorbing features for drop and shock wear (drop and shock wear) and tear experienced in military field applications.

All components that digitally image and detect fluorescence signals exemplarily with rayleigh or raman scattering from the confocal beam's intersection with the device can be integrated into a single structure, such as an ergonomically designed closed structure with a handle that allows it to be held comfortably with one or both hands. The device may also be provided without any handle. The device can be lightweight, portable, and capable of real-time digital imaging and detection of fluorescence signals using rayleigh or raman scattering from a "measurement volume" where any target surface is intersected (e.g., still and/or imaged) using blue or white light fluorescence and/or a reflected imaging mode confocal beam.

By keeping it at a variable distance from the surface, the device can be scanned over the eye tissue surface for imaging and can be used in well-lit environments/chambers to form white or blue light reflectance/fluorescence images. The device can be used in dim or dark environments/rooms to optimize tissue fluorescence signal and minimize background signal from ambient light. The device can be used to visualize ocular tissue (e.g., the lens of the eye) and surrounding tissue (e.g., the retina, vitreous, etc.) either directly (e.g., by the naked eye) or indirectly (e.g., by viewing a digital imaging device screen). The device may have a suitable housing that houses all the components in one piece or integrated as a modular unit to another device, such as a surgical microscope or automated refractor. The housing may be fitted with means to protect any digital imaging device therein. The housing may be designed to be hand-held, small and/or portable. The shell may be one or more sleeves.

An example of a handheld portable device for fluorescence-based monitoring is described below. All examples are provided for illustrative purposes only and are not intended to be limiting. The parameters described in the examples such as wavelength, size and latency may be approximate and are provided as examples only.

In this exemplary embodiment, the device utilizes two violet/blue light (e.g., 430470nm run (run) +/-10 run emission, narrow emission spectrum) LED arrays, each located on either side of the imaging detector assembly, as excitation or illumination light sources. The output power of these arrays was about 1 watt each, diverging from 2.5 x 2.5cm2, with a beam angle of 70 degrees. The LED array may be used to illuminate the ocular tissue surface from a distance of about 10cm, meaning that the total optical power density at the tissue surface is about 0.08W/cm 2. At such low powers, there is no known potential damage to the eye by the excitation light.

The one or more light sources may be articulated (e.g., manually), for example, by using a built-in hinge to vary the illumination angle and spot size on the imaging surface, and powered, for example, by point connections to wall outlets and/or a separate portable rechargeable battery pack. The excitation/illumination light may be generated by sources including, but not limited to: a plurality of Light Emitting Diodes (LEDs) in a single or random arrangement, the arrangement comprising a ring or array form; a wavelength filtering bulb; or a laser. Selected single or multiple excitation/irradiation light sources with specific wavelength characteristics in the Ultraviolet (UV), Visible (VIS), far infrared, Near Infrared (NIR) and Infrared (IR) ranges may also be used, which may consist of LED arrays, organic LEDs, laser diodes or filtered light arranged in various geometries. The excitation/illumination light source may be 'tuned' to allow the intensity of light emanating from the device to be adjusted at the time of imaging. The light intensity is variable. The LED array may be attached to a single cooling fan or heat sink to dissipate heat generated during its operation. The LED array may emit light of any suitable wavelength or wavelengths, which may be spectrally filtered using any suitable commercially available bandpass filter (Chroma Technology Corp, Rockingham, VT, USA) to reduce potential 'leakage' of emitted light into the detector optics. When the device is held adjacent to the eye tissue to be imaged, the illumination source may emit narrow or wide bandwidths of violet/blue wavelengths or other wavelengths or wavelength bands of light onto the surface of the eye tissue, thereby producing a flat image field and a uniform field in the region of interest. The light may also illuminate or excite the tissue down to a shallow depth. The excitation/illumination light interacts with normal and diseased tissue and may cause optical signals (e.g., absorption, fluorescence, and/or reflectance) to be generated within the tissue.

Thus, by varying the excitation and emission wavelengths, the imaging device can interrogate surfaces within the observed ocular tissue structure and ocular tissue components at a depth (e.g., lens, retina, etc.). For example, excitation of a fluorescent source for deeper tissues can be achieved by a change in wavelength from violet/blue (-400-. Similarly, by detecting longer wavelengths, fluorescence emissions can be detected. For medical condition assessment, the ability to interrogate the surface fluorescence of ocular tissue may be useful, for example, in the detection and potential identification of pre-diabetes.

In further embodiments, the device may be used with any standard small digital imaging device, such as a Charge Coupled Device (CCD) or a Complementary Metal Oxide Semiconductor (CMOS) sensor, as an image acquisition device. The example device shown in a) has an external power supply, two LED arrays for illuminating the object/surface to be imaged, and a commercially available digital camera with a stable light device for target acquisition that is ineffective for slight movements by the operator, which is firmly fixed to a lightweight metal frame equipped with a convenient handle for imaging. A multiband filter is held in front of the digital camera to allow wavelength filtering of the detected optical signal emanating from the imaged object/surface. The camera's video/USB output line allows transfer of imaging data to a computer for storage and subsequent analysis. The example embodiment uses a commercially available 8.1 megapixel Sony Digital Camera (Sony Cybershot DSC-T200Digital Camera, Sony corporation, North America). The camera may be suitable because: i) its ultra-thin vertical design (design)n) that can be easily incorporated into a set frame, ii) its large 3.5 inch wide screen touch panel LCD for ease of control, iii) its Carl Zeiss 5 × optical magnifying lens, and iv) its use in low light (e.g., ISO 3200.) devices may have built-in flash, which allows standard white light imaging (e.g., high definition still or video with recorded audio output.) the camera interface end may support wired (e.g., USB) or wireless (e.g., Bluetooth, WiFi, and the like) data transfer or third party add-on modules to various external devices such as a head mounted display, external printer, tablet, palm top computer, personal desktop computer, wireless devices allowing transfer of imaging data to remote stations/other devices, Global Positioning System (GPS) devices, devices allowing use of additional memory and speakers^TMPersonal Digital Assistants (PDAs), and palm top/tablet PCs or personal desktop computers, all of which contain and/or are connected to digital imaging detectors/sensors.

This light signal generated by the excitation/illumination light source may be detected by the imaging device using a filter(s) (e.g., those available from Chroma Technology Corp, Rockingham, VT, USA) that rejects the excitation light but allows emission light of selected wavelengths from the tissue to be detected, thus forming an image or signal on a display as a fluorescence signal or trace. There is a filter holder attached to the casing frame in front of the digital camera lens that can accommodate one or more filters with different discrete spectral bandwidths. These bandpass filters can be selected and arranged in front of the digital camera lens to selectively detect specific optical signals from the surface of the ocular tissue based on the desired wavelengths of light. Spectral filtering of the detected optical signals (e.g., absorption, fluorescence, and reflection) may also be achieved, for example, using a Liquid Crystal Tunable Filter (LCTF) or an acousto-optic tunable filter (AOTF), which is a solid state electronically tunable spectral bandpass filter. Spectral filtering may also include the use of continuously variable filters and/or manual bandpass filters. These devices may be placed in front of an imaging detector to produce multi-spectral, hyper-spectral and/or wavelength selective imaging of tissue.

The device can be modified by using optical or variably oriented polarizing filters (e.g., linear or circular in combination with the use of a light wave plate) connected in a reasonable manner to the excitation/illumination light source and the imaging detector device. In this manner, the device can be used to image tissue surfaces with white light reflectance and/or fluorescence imaging, with polarized light illumination and unpolarized light detection or vice versa or polarized light illumination and polarized light detection. This may allow imaging by minimizing specular reflections (e.g., glints for white light imaging), as well as enabling imaging of fluorescence polarization and/or anisotropy-dependent changes in eye tissue.

In example embodiments, the included devices may also be not handheld or portable, for example, connected to a fixed mechanism (e.g., a tripod or table) to serve as a relatively stationary optical imaging device for white light, fluorescence and reflectance imaging of objects, materials and surfaces (e.g., the eye). This may allow the device to be used on a table or for "assembly line" imaging of objects, materials and surfaces. In some embodiments, the securing mechanism is movable.

Other features of the device may include the ability to record digital images and video, possibly with audio, documentation methods (e.g., via image storage analysis software) and wired or wireless data transmission for remote medical/electronic health needs. For example, embodiments of the device are configured to include a mobile communication device such as a mobile phone. The mobile phone used in this example is of the Samsung a-900 type, which is equipped with a 1.3 megapixel digital camera. The phone is mounted to a cradle to facilitate imaging. The images from the mobile phone may be sent wirelessly to another mobile phone or wirelessly (e.g., via bluetooth) to a personal computer for image storage and analysis. This indicates that the device performs real-time handheld fluoroscopic imaging and wireless transmission to the patient as a remote medical/electronic healthRemote station/personal ability of the diabetes care infrastructure portion. To demonstrate the ability of the imaging device in healthcare and other related applications, some feasibility tests were performed using the specific examples described. It should be noted that during all fluorescence imaging experiments, the Sony Camera (Sony Cybershot DSC-T200Digital Camera, Sony corporation, North America) settings were set to use the 'Macro' imaging mode setting without flash capturing images. The image was captured at 8 megapixels. A white light reflectance image is captured using a flash lamp. All images are stored on xD memory cards for subsequent transfer to personal computers for long term storage and image analysis. All white light reflectance and fluorescence images/movies captured by the device were input to Adobe Photoshop for image analysis. However, using MatLab^Tm(Mathworks) image analysis software was designed to allow for the use of various image-based spectral algorithms (e.g., red to green fluorescence ratio, etc.) to extract relevant image data (e.g., spatial and spectral data) for quantitative detection/diagnostic value. Image post-processing also includes mathematical operations of the image.

Further, an improved fixation target system is provided that advantageously applies visual cues through the alignment tube to help the patient self-align and locate the holes to help the patient more easily determine where to move his head and/or eyes (gaze) to achieve alignment with the LED fixation target, which may or may not blink. In one aspect, the alignment tube comprises a cylinder whose interior cavity surface is shiny or highly reflective along its length. Centrally located at the distal end of the cylindrical tube is an LED assembly. For example, a suitable cylinder is metal or may be plastic or any other material as long as the interior cavity is shiny and highly reflective of the cylindrical surface. See fig. 6 (photo) and 6A (schematic) showing an example embodiment comprising a metal tube with LEDs and holes embedded in the ends of the tube, of which the leads are shown.

This enables the patient to see the inner wall of the tube illuminated by the LEDs at a considerable angle so that he can easily see the tube entrance and try to align his field of view by moving his head to the centre of the set of nested circles formed by the multiple reflection images of the LEDs. When the patient looks through the line of sight along the tube axis, the ferrule will appear concentric and centered on the LED. See fig. 7 (schematic) and 7A (photograph). When the patient's field of view is misaligned, i.e., not properly along the central axis of the lumen, the circles will not appear concentric (i.e., skewed from the axis), and the LED target may not be directly visible. See fig. 8 (schematic) and 8A (photograph). The patient can then self-align by making body, head or eye adjustments to center the LED in the reflective circle and finally center the LED target along, for example, the center axis of the LED gaze alignment tube.

In a further embodiment, the LED fixation aiming tube may be translucent and backlit along its length by a series of alternating opaque and transparent annular rings by light sources of different colors. The patient's field of view will be similar to that described above with respect to the eccentric ring seen when the lines of sight are not aligned. See fig. 9, which depicts a field of view of a patient centered on the exemplary optical axis of this example embodiment.

In other embodiments, a fixation point such as a flashing LED may be used without a tube to position the subject's eye, as described above, to align the subject's eye so as to present a precise inferior position to which the excitation light is directed. The gaze points are provided by LEDs of any suitable color, which may include gaze targets in one or more of the gaze points in a cross-hair configuration to assist the gaze of the subject's eyes. In an alternative embodiment, it is envisioned that the point of regard may be in optical communication with a beamsplitter disposed at a suitable angle of incidence with respect to the subject's eye, and may reflect the point of regard into the subject's eye. In a computer automated system, the subject eye must be fixated before the excitation light is directed to the subject eye and the sample volume is collected.

Other example embodiments include apparatus and methods adapted to determine properties of in vivo tissue by collecting spectral information from the lens of the eye. The illumination light system provides excitation light of one or more wavelength ranges that is in communication with an optical collection device (e.g., a light detector). In some embodiments, a light homogenizer and an mode scrambler may be used to modify performance. The optical system is non-invasive and, moreover, does not physically contact or invade the eye or skin. The optical source primarily receives light from the illumination system and emits it to the lens of the eye. An optical collection system and/or device receives light emitted from the eye's lens tissue in response to excitation light by its fluorescence. The optical collection system may communicate the light to a spectrometer that generates a signal representative of a spectral property of the light. The analysis system (computer) determines the properties of the eye lens from the spectral properties.

In further embodiments, methods are provided for determining a measure of a tissue or disease state (e.g., glycation end-product or disease state) in an individual. A portion of the individual tissue is illuminated with excitation light and light emitted by the tissue is detected due to fluorescence of chemicals in the tissue in response to the excitation light. The detected light may be combined with a model that relates fluorescence to a measurement of tissue state to determine the tissue state. Embodiments may include single wavelength excitation light, scanning excitation light (illuminating tissue at multiple wavelengths), detecting at a single wavelength, scanning detection wavelength (detecting emission light at multiple wavelengths), and combinations thereof. Example embodiments may also include calibration techniques that reduce measurement errors due to the detection of light rather than the detection of fluorescence of chemicals in tissue. For example, the reflection of tissue can cause errors if appropriate corrections are not applied. Embodiments may also include various models relating fluorescence to tissue state measurements, including various methods for generating such models. Other biological information is used in conjunction with the fluorescence properties to aid in the determination of the tissue state. Embodiments also include apparatus adapted to perform the method, including a suitable light source, detector, and model (e.g., implemented on a computer) for correlating detected fluorescence with tissue state measurements.

Some example embodiments provide techniques for measuring light scattering in a subject's eye, such as a human eye, for diagnostic purposes. For example, the light scattering system includes an excitation light assembly that emits light (e.g., an LED or laser beam) into the subject's eye. The transmission lens focuses the scattered laser light to form an image on the measurement mirror. Between the transmission lens and the measuring mirror, the light is reflected from a controllable mirror, which can be adjusted to position the image at a desired position on the measuring mirror. The pinhole of the measurement mirror allows some of the scattered laser light to pass through and be detected by a single photon detector and analyzed by a hardware or software correlator. The scattered laser light that does not pass through the pinhole is reflected by the measuring mirror towards a charge-coupled device (CCD) camera. The camera obtains an image of the scattered laser light and provides the image to a computer. The computer obtains information from the correlator and an image from the camera. The computer may analyze the output of the correlator (correlation function) which correlates the measured scattered light with the location within the eye to determine if the eye has an indication of an abnormality, such as a disease. The computer may also process image information from the camera to provide an image of scattered light from the eye and send control signals to the deflection mirror to adjust the movement of the subject's eye and to help ensure that light from the desired location of the eye is directed through the measurement mirror pinhole. However, this light scattering system is exemplary, not limiting, as other implementations are possible in accordance with the present disclosure.

In further embodiments, an excitation light source (e.g., a blue LED) may be used to illuminate a particular point in the subject's lens of the eye that is about 50% to 80% (optionally 60% to 75%) from the leading edge of the subject's lower quadrant of the lens, and all subranges therebetween. It has been determined that measurements at this location in the mammalian eye provide consistent measurements without undesirable delays or interference that would misinterpret data collection. For example, care should be taken in fluorescence spectroscopy to avoid confounding the effects of undesired optical signals in the detection of compounds of interest (e.g., AGEs). The effects of macular pigment, cataracts, fluorescence emissions, etc. from areas outside the lens can potentially be confounded. The effects of these and other factors can be reduced by selecting an excitation wavelength that is well outside the absorption of the undesired effects, but still overlaps with the AGE absorption in the green wavelength region on its long wavelength shoulder. Thus, as mentioned above, measurements from the lower position are advantageous, since interference is minimized.

In an example embodiment, the returned light may include fluorescence generated by AGEs in the lens of the eye. The intensity of the returned fluorescence can be compared to an age-related expected fluorescence intensity for an individual who does not have diabetes. Optionally, the amount by which the intensity of the actual returned fluorescence exceeds the intensity of the expected returned fluorescence can then be used to determine the duration and/or severity of the individual experiencing the medical condition. Temporal characteristics of the fluorescence rather than intensity can also be detected and used to determine how long the patient has experienced the medical condition. Temporal characteristics may be analyzed by any suitable technique including, but not limited to, direct measurement of decay time of the fluorescence emission, by a phase shift technique, by a polarization anisotropy technique, or by any other method of detecting temporal characteristics of fluorescence.

In still other example embodiments of the present invention, the returned light may include backscattered excitation light, which is returned from the target tissue. Such embodiments may utilize only backscattered light for determination, or backscattered light may be used in conjunction with fluorescence generated by the target tissue to effect determination. In some of the present embodiments, a light source providing excitation light and a detector detecting returned light are arranged as a confocal system. As previously described, such confocal systems allow interrogation of small target tissue volumes within a larger tissue volume. Confocal systems allow measurements to be made on a volume of tissue below the surface of the target tissue. Also, in example embodiments, the system or method may also take into account patient-specific information. For example, in addition to optical information, patient age, gender, and other desired physical characteristics may be applied in various combinations to determine how long a patient has experienced a medical condition. This would allow the system or method to account for age-changing characteristics, such as fluorescence intensity.

The excitation light source may be a laser, LED, fluorescent tube, incandescent bulb, halogen or arc lamp, or any other type of device capable of providing excitation light in the appropriate wavelength range. The light source may also include a broadband light source such as a fluorescent or incandescent light bulb. Such a broadband light source may also be paired with one or more filters designed to pass only light of a particular wavelength band. The light source may also comprise any other type of light source depending on the wavelength of interest. For example, the excitation light source may be a He-Cd or argon ion laser, a mercury lamp, a low power white or blue LED, or the like. The excitation filter may be, for example, a long or short bandpass filter having a suitable wavelength. The excitation filter may be selected to attenuate wavelengths that do not correspond to the excitation wavelength. The filtered light may then be directed to a dichroic mirror, such as a long pass dichroic mirror, to be redirected toward the lens.

In order to measure fluorescence and backscatter data quickly enough to calculate the fluorescence to backscatter ratio, a spin filter disk or a fast-changing monochromator may be used and located at a point in the optical light path in front of the light detector. In certain example embodiments, the spin filter wheel includes a circular filter array having a pattern of four filter elements or materials that allow transmission of alternating wavelengths at different rotational positions around the circular filter array. The filter array may be rotated by the motor to discrete angular positions. The system for repeatedly returning to the desired angular position can be provided by a dial or by a memory element connected to the motorized system. Some examples of motorized systems are stepper motors, which are capable of initializing angular positions, or servo motors with encoders that provide initialization information. By using a spin filter disk, more data points can be collected and averaged to obtain a near real-time data set. This is achievable because each data measurement takes less than 30 seconds each (as in the Accu-Chek D-controller). The filter selection by continuously rotating a filter wheel directly coupled to the stepper motor shaft allows for rapid (i.e., several cycles per second) filter changes. Specifically, two pairs of blue (to measure rayleigh backscattering) and green (to measure fluorescence) filters are positioned alternately around the disk surface. The use of 4 filters allows the use of a small, lower cost circular filter instead of two larger custom semicircular filters. By detecting a coded notch along the periphery of the edge of the filter disc with a pair of photo-interrupters or the like, it is determined which filter is in the optical path in front of the photo-detector.

In operation, a first scan along the optical axis takes less than 1.5 seconds to measure the position of the anterior and posterior faces of the lens, followed by a second scan. During the second scan, the filter wheel is rotated sufficiently 4 times per second (i.e., once every 0.25 seconds) to provide 16 filter changes per second. For example, a total of 50 readings may be taken per filter every 0.25 seconds. Thus, the skilled artisan will appreciate that the use of a spin filter disk is a significant improvement over the filter sliding mechanism employed by the Accu-Chek D-Tector. In particular, the collection of two sample volumes may be consistently achieved in ten (10) seconds or less, and in some instances eight (8) seconds or less.

The following is a schematic description of an example embodiment of an apparatus comprising a confocal configuration (note that unlike previous apparatus, the light path does not encounter any dichroic mirror or dichroic mirror, thereby increasing the energy of light transmission):

alternatively, in another exemplary embodiment, moving parts required due to the presence of the spin filter disk, which are susceptible to periodic mechanical maintenance to prevent malfunction, may be eliminated by a light detection system that employs a dichroic beam splitter (or dichroic mirror) and two light detectors, whereby light having a wavelength greater than 500nm is reflected by the beam splitter to a first detector while light having a wavelength less than 500nm is transmitted through the beam splitter to a second light detector. The advantage of this configuration is that there are no moving parts and no defined time in reading the two channels, since both photodetectors are collecting data 100% of the time. In fluorescence microscopes, a dichroic mirror separates the light paths. In other words, the excitation light reflects the dichroic mirror surface into the light detector. The fluorescent emission passes through a dichroic mirror to a light detection system. As mentioned above, the specific reflective properties inherent in the dichroic mirror allow it to separate two wavelengths-called shifted wavelength values, which are wavelengths with 50% transmission. The dichroic mirror reflects wavelengths of light having a shift wavelength value or less and transmits wavelengths having a wavelength value or more. Ideally, the wavelength of the dichroic mirror is chosen to be between the wavelengths used for excitation and emission. However, about 90% of the light with a wavelength below the shifted wavelength value is reflected by the dichroic mirror and about 90% of the light with a wavelength above this value is transmitted. When the excitation light illuminates the eye lens, a small amount of the excitation light is reflected off of the optical elements within the objective lens, and some of the excitation light is scattered back through the sample to the objective lens. Some of this excitation light is transmitted through the dichroic mirror along with the longer wavelength light emitted by the sample. By using wavelength selective elements such as emission filters, this "contaminated" light can be prevented from reaching the detection system.

In an exemplary embodiment, two filters are used with a dichroic mirror. The excitation filter can be used to select the excitation wavelength by placing the excitation filter in the excitation path just before the dichroic mirror. Because by placing it below the dichroic mirror, the emission filter can be used to more specifically select the emission wavelength of light emitted from the lens of the eye and remove traces of excitation light. In this position, the filter serves both to select the emission wavelength and to eliminate any traces of the wavelength used for excitation. These filters are often referred to as interference filters because of the way they block the transmission of the bands. The interference filter shows extremely low transmission outside its characteristic band pass. They are therefore suitable for selecting the desired excitation and emission wavelengths.

Another alternative derives from the observation that: the intensity of the blue (scattered) signal is about 4 times higher than the green (fluorescent) signal. Using a 75%/25% beamsplitter with a green bandpass filter for the 25% path and a blue filter for the other path will produce signals from both detectors of about the same magnitude. A further alternative could be the use of a grating or linear variable filter wavelength dispersive element in front of the linear array light detector. Another embodiment may be the use of an electronically adjustable bandpass filter (such as a piezo-controlled wavemeter) in front of a single light detector. A further alternative may be to alternate between the two filters (blue and green) by moving the filters using the oscillatory motion of a resonant mechanical oscillator, such as a tuning fork. Furthermore, if only a green signal is desired, it can be measured with a single detector and green filter.

In an embodiment, a blue LED light source generates excitation light that is coupled to one or more optical bandpass filters to generate excitation light having a desired wavelength. The excitation radiation in the appropriate wavelength band is then directed through an optical delivery system that focuses the excitation light on the target tissue in the patient's eye. The returning light, which may include backscattered portions of the excitation light and/or fluorescence generated in response to the excitation light, is then collected by a light detector for analysis. One or more excitation wavelengths may be used and one or more fluorescence wavelengths may be converged.

In an example embodiment, the blue LED light source is an integrated assembly comprising a high intensity (18,000mcd)465nmInGaN LED with a 15 degree viewing angle in a molded 3mm diameter transparent lens-fitted package. This low cost, long life light source replaces the expensive, laser-based frequency doubled 473nm light source in the Accu-Chek D-tester. It should be appreciated that the elimination of a laser as the light source eliminates the need for an undesirable laser safety subsystem. The integrated assembly also includes a 1mm diameter hole, which is arranged almost in contact with the LED lens. For example, with a conical hole, the thickness of the hole can be minimized to eliminate reflections from the hole ID. An optical bandpass filter may be used to block the observed spectral tail of the blue LED emission. For example, a 58nm wide bandpass filter centered at 450nm, 2.0 optical density, which blocks light outside the band, may be applied. Further, by moving the base horizontally within the confines of the slot for fitting the screw into the light fixture plate, the positional adjustment of the blue LED light source assembly can be roughly adjusted laterally. The horizontal and vertical source positions are fine-tuned by means of a bent fitting structure which is adjusted and clamped by a push-pull screw pair. It should be understood that additional light intensity may be obtained by using an optional LED source converging lens, whereby the light from the aperture LED includes a diverging cone that fills the source lens. The addition of a converging lens after the aperture would shrink the cone angle to just fill the source lens, thereby producing more light in the source beam.

In an embodiment, a blue LED light source generates excitation light that is coupled to one or more optical bandpass filters to generate excitation light having a desired wavelength. The excitation radiation in the appropriate wavelength band is then directed through an optical delivery system that focuses the excitation light on the target tissue in the patient's eye. The returning light, which may include backscattered portions of the excitation light and/or fluorescence generated in response to the excitation light, is then collected by a light detector for analysis. An example implementation of an LED light source assembly is described in fig. 10.

In the example embodiment described above, the blue LED light source is an integrated assembly comprising a high intensity (18,000mcd)465nm InGaN LED having a 15 degree viewing angle in a molded 3mm diameter package with a transparent lens. This low cost, long life light source replaces the expensive, laser-based frequency doubled 473nm light source in the Accu-Chek D-tester. It should be appreciated that the elimination of a laser as the light source eliminates the need for an undesirable laser safety subsystem. The integrated assembly also includes a 1mm diameter hole, which is arranged almost in contact with the LED lens. For example, with a conical hole, the thickness of the hole can be minimized to eliminate reflections from the hole ID. An optical bandpass filter may be used to block the observed spectral tail of the blue LED emission. For example, a 58nm wide bandpass filter centered at 450nm, 2.0 optical density, which blocks light outside the band, may be applied. Further, by moving the base horizontally within the confines of the slot for fitting the screw into the light fixture plate, the positional adjustment of the blue LED light source assembly can be roughly adjusted laterally. The horizontal and vertical source positions are fine-tuned by means of a bent fitting structure which is adjusted and clamped by a push-pull screw pair. It should be understood that additional light intensity may be obtained by using an optional LED source converging lens, whereby the light from the aperture LED includes a diverging cone that fills the source lens. The addition of a converging lens after the aperture would shrink the cone angle to just fill the source lens, thereby producing more light in the source beam.

With any number of suitable algorithms, pupil tracking helps maintain eye alignment and compensate for slight head or eye movement so that appropriate illumination can be provided to the eye for accurate imaging/data collection. Due to the near real-time nature of facilitating data acquisition through the use of the spin filter wheel of an embodiment, the need to use a pupil tracker and its associated software/hardware to monitor eye movement and patient alignment during data acquisition is virtually eliminated. Such a pupil tracking system may be implemented using any suitable imaging and/or coordinate tracking device in an external coordinate system that may be used to track the position of a body region, such as a patient's eye. In the case of tracking a patient's eye by determining the position of the geometric axis of the eye, the tracking system may include (i) a camera for imaging the tracked body region, (ii) a light source (e.g., an infrared light source) illuminating the imaged region, and (iii) a detector on which the camera image may be represented as a digital image. Suitable tracking systems may include imaging and signal responsive elements. Standard or commercially available imaging and image-processing system components may be adapted and applied.

The body of the instrument can be sealed to prevent ambient light from adversely affecting data collection through the confocal spectroscopy device housed therein. In essence, the patient's head is restrained by adjustable forehead and jaw rests. The curvature of the forehead rest is formed to be less than the curvature of most human forehead rests to ensure stable two-point contact with the forehead at each end of the forehead rest. The forehead rest is manually adjustable into and out of the instrument to move the patient's head to accommodate the patient's eye socket depth. The motorized jaw rest is vertically movable up and down to accommodate the length of the patient's head and may be controlled by on-screen buttons on the operator's computer or other suitable means. The flexible contoured sight cup serves to block ambient light inside the instrument. The sight cup may be slightly compressed by the patient to ensure a light seal. The horizontal position of the sight cup is adjustable via an operator controlled motor to accommodate the patient with different interpupillary distances (PD's) to ensure that the instrument is positioned horizontally from the forehead/jaw centerline for proper viewing of the patient's eyes.

The sight cup may be disposable or permanently affixed so long as it is configured to contact the patient's eye socket to substantially block ambient light and/or at least partially support the body on the user's eye socket. The sight cup has a central opening/aperture to allow light to pass from an excitation light source housed in the body to the patient's eye. The sight cup may be made of paper, cardboard, plastic, silicon, rubber, latex, or a combination thereof. The sight cup may be a tubular, conical, or cup-shaped flexible or semi-rigid structure with an opening on either end. Other materials, shapes, and designs are possible so long as the surrounding light is not allowed to pass through the interface between the viewing cup and the patient's eye socket. In some example embodiments, the optic cup is made of latex rubber that conforms around the ocular (eyepiece) portion of the subject and is compressible (as shown). Optionally, the sight glass may be detached from the body after the eye scan has been completed, and a new sight glass may be attached to the new user to ensure hygiene and/or prevent disease transmission. The sight cup may be transparent, translucent, or opaque, although opaque sight cups provide the advantage of blocking ambient light for measurements made in well-lit environments. While the body may include one or more sight cups, which may be oriented in a binocular manner, only one sight cup is necessary to measure AGE, thereby keeping manufacturing costs low. In an alternative embodiment, the sight cup may be removed in the event that the instrument is operated in a medium to low ambient lighting environment. Additionally, instead of a sight cup, an anti-reflective coated window disposed at the eye opening prevents airflow through the patient's eye and helps to minimize dust accumulation on the internal light devices of the system. The window is tilted to avoid specular reflections back to the photodetector.

In the illustrated example, the subject is a monocular system configured to scan one eye without repositioning the oculars (oculars) with respect to the patient's head, thereby reducing the time to scan the patient. Optionally, the subject may include a binocular or binocular system or optical paths to both eyes for eye scanning (e.g., two eyepieces or optical paths for both eyes of the patient, one field of view for one eye and the other field of view for the other eye, etc.), whereby both eyes are scanned simultaneously, which provides interlaced measurements of both eyes. Other embodiments are also possible, for example, a binocular system or a two eye lens system, with two respective optical paths to each eye, may be configured to scan consecutive eyes, meaning first eye and then second eye. In some embodiments, continuously scanning the eye comprises scanning a first portion of the first eye, a first portion of the second eye, a second portion of the first eye, and so on.

Other methods are also possible. In some example embodiments, for example, the body includes a jaw rest that may be configured to automatically adjust or allow manual adjustment between the body (and/or the sight cup) and the patient's eye. The adjustment may be fine, on the order of about 0.5, 1, 2, 3, 4, 5, 10, 20, 30, or 50 millimeters. The adjustment may include any of the adjustments described herein, such as adjusting one or more movable optical components, for example, to improve the field of view. In one example, the distance between the subject and/or an optical component in the subject and the patient's eye is adjusted to a second distance from the first distance system. In some embodiments, the jaw rest is movable, although in various embodiments the jaw rest may be fixed while other components within the body are movable. The distance may be based at least in part on a normalized value such as an average offset between the jaw and the pupil (e.g., in an anterior-posterior direction) or an average distance between the pupil and the optic cup. In some examples, the distance may be determined based at least in part on the sensor readings. For example, the sensor may detect the position of the user's eye, pupil, or iris. The sensor may comprise an optical or ultrasonic instrument. For example, the sensor may emit light and determine the time elapsed between emissions and the time at which reflected light (e.g., pulses) is received. The sensor may comprise a weight sensor to detect, for example, the position of the patient's jaw. The sensor may detect the position or weight of the jaw of the user. In certain example embodiments, the jaw rest is movable or the body and/or eyepiece/sight cup of the body are moved relative to the jaw rest and the field of view is monitored as described above to determine the appropriate position of the eye. Other variations are also possible.

In some embodiments, the position/setting of one or more movable/adjustable optical components may be manually adjusted by the patient. The patient may be instructed to adjust the position/setting, for example, based on one or more images seen by the patient. For example, the patient may be instructed to adjust the position until two or more images (e.g., working distance images) are aligned. The arrangement may correspond to an appropriate distance of the eye to the instrument. Other designs are also possible.

With each filter in the filter wheel aligned, circuitry is operatively connected to the photodetector to sample the signal intensity. The circuitry is controlled by a computer program to generate spectral data and information from the sample data. The implementation of such control and measurement circuits is known to those skilled in the art. For example, in an example embodiment, a computer system (not shown) is electrically connected to the output device and the communication medium. Communication media may enable the computer system to communicate with other remote systems. A computer system is electrically connectable to the body for collecting data and analyzing the data according to an algorithm. Optionally, the sight cup and jaw rest motor may be configured to be controlled by the computer system to semi-automatically position the sight cup and jaw rest to match the interpupillary distance between the user/patient's eyes. In these examples, the eye tracking device may include the system described herein. In various embodiments, the above-described combinations are used to adjust the distance of the sight cup relative to the chin rest and/or headrest to match or substantially conform to the user's interpupillary distance.

The interpupillary distance may be adjusted based on the patient's view of the fixation target. For example, the fixation target may be configured to require the user to align the fixation target with a suitable alignment means. A red LED may be used as one example implementation of a fixation target; however, other fixation targets are possible including, but not limited to, a box configuration or two or more LEDs, etc.

Thus, in an example embodiment, the system described herein may include software configured to determine the output of the eye and/or compare the measurement to other previously taken measurements (e.g., previously obtained measurements from the patient or baseline measurements). The software may be located at a remote location, such as a server. The raw image data or the extracted digital data may be transmitted to a remote location, such as a server, and calculations and/or comparisons made at the remote location. In some embodiments, data corresponding to a prior test need not be sent to the system, for example, where the comparison is made at a remote location, such as a server. In some embodiments, the analysis is performed both at the subject location and at a remote location, such as a server. Thus, suitable software may be included in both the subject and the remote location. The output may include a probability such as a probability of a condition deteriorating or improving. The output may include a confidence measure. As another example, the output may indicate that the eye condition is worsening, improving, or substantially unchanged. The output may include a reservation request. For example, if it is determined based on the obtained data that a particular change has occurred or that a threshold has been crossed, an output including a reservation request can be sent to the healthcare provider. The output may also include an indication of a recommendation for consultation (referral) or reservation or other follow-up activity.

In general, in another aspect, example embodiments provide a system that performs at least one of light scattering and fluorescence scanning of a subject eye, including a display screen displaying an image of the eye to allow an operator to select a position in the eye to be measured. The system may include an optical unit connected to the processor for performing a scan at a selected location of the eye and for collecting data relating to the detected light scatter and/or fluorescence. The processor may further display the data on a display screen for viewing by an operator. To this end, data may be reported on the same display screen and/or collected in a loop. Further, the data displayed on the display screen may include test settings, front and cross-sectional views of the eye, average intensity values of detected light scatter and/or fluorescence, graphical descriptions of the autocorrelation function, and curve fitting parameters based on an exponential fit to the autocorrelation data. The data can be used to detect the presence of material or objects of interest, including but not limited to AGE and/or disease tracking progression.

In some embodimentsThe collected data may include the average intensity of the detected scattered light and/or the average intensity of the detected fluorescence. Implementations of this embodiment may also collect data from locations in the nucleus and/or supranucleus region of the ocular lens to determine a ratio of the mean fluorescence intensity associated with the fluorescent ligand scan of the nucleus region of the ocular lens to the mean fluorescence intensity associated with the fluorescent ligand scan of the supranucleus region of the ocular lens. A similar ratio can be determined for quasi-elastic light scattering in the nucleus and supranuclear regions of the ocular lens. The ratio may be related to a disease state in the eye such that an increase in the ratio indicates an increase in the amount of material and/or object in the eye. Some embodiments may also incorporate a measurement quality metric that is calculated as follows: by multiplying these ratios together or using a curve, y (t) ═ Le^-ktWhere I is the mean intensity, k is the decay time constant and t is the time. Additional system aspects may include a display screen for displaying images to allow an operator to select an eye region for analysis and a program configured to analyze the light scattered from the quasi-elastic light scatter and/or the fluorescent emissions from the fluorescent ligand scan to detect a material or object of interest located within the selected eye region. The material or object of interest may be, but is not limited to, an AGE. In some embodiments, the average intensity of scattered and/or fluorescent emissions from the nuclear and/or nuclear region of the ocular lens may be analyzed. Further, the average intensity of scattered light or fluorescent emission from the nuclear region of the ocular lens may be compared to the average intensity of scattered light or fluorescent emission from the supranuclear region of the ocular lens to provide a correlation factor for calculating the presence of a material or object of interest in the eye. In other example embodiments, the processor may measure the fluorescence intensity from the region of the eye before introducing the imaging agent and after introducing the imaging agent to determine the difference between the two intensities. In some embodiments, the processor may measure first data of eye fluorescence prior to introducing the imaging agent into the eye and second data of eye fluorescence after introducing the imaging agent into the eye, and then compare the first data and the second data. The comparison may include, for example, subtracting the first data from the second data to determine a difference in the measured fluorescence. In addition, processThe display may display data from the quasi-elastic light scattering and/or fluorescent ligand scans on a display screen for viewing by an operator. The data may include any information about the quasi-elastic light scattering and/or the fluorescent ligand scanning performed.

Further, another exemplary embodiment is an apparatus, comprising: an excitation light source adapted to excite AGE autofluorescence, optionally a filter to remove undesired wavelengths, and a photodetector coupled to the filter to detect an eye tissue (e.g., retinal tissue) fluorescence signal generated in response to the excitation light, producing a signal indicative of the integrated intensity of the eye tissue fluorescence signal; optionally, a photon enhancer connected to the photodetector to enhance the eye tissue fluorescence signal; and a computing device communicatively connected to the light detector, the computing device configured to generate one or more of the following based on the signal indicative of the integrated intensity: an indication of whether the patient has diabetes (e.g., overt diabetes, pre-diabetes, gestational diabetes, etc.), an indication of whether the patient has an ocular condition resulting from diabetes, an indication of whether the patient has serous central retinopathy, an indication of whether the patient has diabetic retinopathy, an indication of whether the patient has retinal vessel occlusion, an indication of whether the patient has vitreoretinopathy, an indication of whether the patient has any other acquired retinopathy, an indication of whether the patient has age-related macular degeneration, an indication of whether the patient has hereditary retinal degeneration, an indication of whether the patient has pseudotumor cerebri, an indication of whether the patient has glaucoma.

The advantages of the example embodiments may be realized and attained by means of the instrumentalities and combinations particularly pointed out in this written description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

While example embodiments have been illustrated in detail, the foregoing description is in all aspects illustrative and not restrictive. It should be understood that numerous other modifications and variations can be devised without departing from the scope of the example embodiments.

While example embodiments have been described in connection with what is presently considered to be practical for the intended purpose, it is to be understood that the description is not limited to the particular embodiments disclosed, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of example embodiments. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific example embodiments specifically disclosed herein. Such equivalents are intended to be encompassed within the scope of the claims appended hereto or as subsequently filed.

Claims (11)

1. Apparatus for determining a characteristic of a biological tissue of a subject, comprising:

a) means for irradiating said biological tissue with electromagnetic radiation directed into said biological tissue, said means for irradiating comprising a blue LED light source, thereby causing said biological tissue to react with a first effect selected from the group consisting of reflection, backscattering, transmission and emission of responsive radiation and a second effect selected from the same group excluding the first effect;

b) a detector that converges the responsive radiation;

c) a filter coupled to the detector for separating the concentrated radiation into a plurality of components;

d) a processor operable to (i) measure an intensity of each of the separated plurality of components and (ii) determine a mathematical relationship between the separated plurality of components;

e) an LED holding tube for guiding the object to self-align along an axis;

f) an aperture located on the light source;

g) a lens optically responsive to light from the light source after passing through the aperture for focusing the light; and

h) a lens system optically responsive to the focused light.

2. The apparatus of claim 1, wherein said means for irradiating causes said biological tissue to react by emitting responsive radiation, further comprising means for measuring a time difference between irradiating said biological tissue and emitting said responsive radiation.

3. Apparatus for measuring molecular changes in a patient having an ocular lens capable of backscattering radiation when illuminated, the radiation including fluorescent and rayleigh components of detectable intensity, comprising:

a) means for illuminating said ocular lens with light having a wavelength between 465-500nm resulting in backscattered radiation in response to said illumination, said means for illuminating comprising a blue LED light source;

b) a detector responsive to said backscattered radiation for converging said backscattered radiation;

c) a filter for separating the backscattered radiation into a fluorescent component and a rayleigh component;

d) a processor operable to (i) detect the respective intensities of the separated fluorescent and rayleigh components and (ii) calculate a detected intensity ratio, thereby producing a measurement of molecular changes in the ocular lens; and

e) an LED fixation tube for guiding the patient to self-align along an axis;

f) an aperture located on the light source;

g) a lens optically responsive to light from the light source after passing through the aperture for focusing the light; and

h) a lens system optically responsive to the focused light and defining an aperture at a focal point thereof greater than 15 microns.

4. The apparatus of claim 3, further comprising ocular means responsive to said backscattered radiation for allowing an operator to view said ocular lens.

5. The apparatus of claim 4, wherein said filter comprises at least one dichroic beamsplitter.

6. The apparatus of claim 5, wherein the detector comprises at least one single chip silicon detector.

7. The apparatus of claim 6, further comprising an amplifier.

8. The apparatus of claim 7, wherein said means for illuminating is operable to adjust a power level of said light source.

9. Apparatus for measuring molecular changes in a patient having an ocular lens capable of backscattering radiation when illuminated, the radiation including fluorescent and rayleigh components of detectable intensity, comprising:

a) a blue LED for providing light having a selected power level and wavelength selected between 465-500nm, wherein the power level is adjustable in response to the provided light;

b) an aperture located on the light source;

c) a lens optically connected to the adjusting means for focusing the light after passing through the aperture;

d) a first optical fiber optically coupled to the lens for receiving the focused light;

e) a lens system optically connected to said first optical fiber and defining an aperture having a focal point greater than 15 microns for delivering said focused light to a selected volume of said ocular lens of about 200 microns, thereby causing backscatter radiation in response to said delivered light;

f) a light collector, (i) having a focal point surrounding a selected volume of the ocular lens to which the focused light is delivered and (ii) responsive to the backscattered radiation for concentrating the backscattered radiation;

g) a second optical fiber optically connected to the collector for receiving the concentrated radiation;

h) a filter for separating the backscattered radiation into its fluorescent and rayleigh components; and

i) a processor operable to (i) detect respective intensities of the separated fluorescent and rayleigh components, and (ii) calculate a detected intensity ratio, thereby producing a measurement of molecular changes in the optical lens; and

j) an LED mounting tube for guiding the patient to self-align along an axis.

10. The apparatus of claim 9, wherein the diameter of the aperture is about 1 mm.

11. The apparatus of claim 1, wherein the diameter of the aperture is about 1 mm.