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WO1997008523A1 - Procede et appareil de determination des caracteristiques d'un echantillon en presence de lumiere ambiante - Google Patents

Procede et appareil de determination des caracteristiques d'un echantillon en presence de lumiere ambiante Download PDF

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Publication number
WO1997008523A1
WO1997008523A1 PCT/US1996/013523 US9613523W WO9708523A1 WO 1997008523 A1 WO1997008523 A1 WO 1997008523A1 US 9613523 W US9613523 W US 9613523W WO 9708523 A1 WO9708523 A1 WO 9708523A1
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WIPO (PCT)
Prior art keywords
sample
light
light intensity
detector
ambient light
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1996/013523
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English (en)
Inventor
Glenn F. Spaulding
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Abraxis Bioscience LLC
Original Assignee
Vivorx Pharmaceuticals Inc
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Filing date
Publication date
Application filed by Vivorx Pharmaceuticals Inc filed Critical Vivorx Pharmaceuticals Inc
Priority to AU68536/96A priority Critical patent/AU738290B2/en
Priority to JP9510404A priority patent/JPH11511557A/ja
Priority to EP96928966A priority patent/EP0866953A4/fr
Publication of WO1997008523A1 publication Critical patent/WO1997008523A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J1/4204—Photometry, e.g. photographic exposure meter using electric radiation detectors with determination of ambient light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J1/44—Electric circuits
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/10—Arrangements of light sources specially adapted for spectrometry or colorimetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/42—Absorption spectrometry; Double beam spectrometry; Flicker spectrometry; Reflection spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/46—Measurement of colour; Colour measuring devices, e.g. colorimeters
    • G01J3/50—Measurement of colour; Colour measuring devices, e.g. colorimeters using electric radiation detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/46—Measurement of colour; Colour measuring devices, e.g. colorimeters
    • G01J3/50—Measurement of colour; Colour measuring devices, e.g. colorimeters using electric radiation detectors
    • G01J3/501—Colorimeters using spectrally-selective light sources, e.g. LEDs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/46—Measurement of colour; Colour measuring devices, e.g. colorimeters
    • G01J3/52—Measurement of colour; Colour measuring devices, e.g. colorimeters using colour charts
    • G01J3/524—Calibration of colorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/42—Photometry, e.g. photographic exposure meter using electric radiation detectors
    • G01J2001/4242—Modulated light, e.g. for synchronizing source and detector circuit
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G01N2201/124—Sensitivity
    • G01N2201/1244—Ambient light detector, e.g. for invalidating

Definitions

  • the present invention relates generally to processes for detecting the intensity of light emanating from a sample in the presence of ambient light and, is useful in determining colorimetric or fluorescent light intensity of a sample, or the amount of a particulate in a sample based upon the detected intensity when the intensity is measured under ambient lighting conditions. Such light intensity measurements are useful in determining a large variety of properties of a large variety of samples.
  • Microscopic and macroscopic particles contained in a sample can diffract, reflect, transmit, scatter, fluoresce, phosphoresce, or absorb light.
  • the emitted light is typically of a different wavelength than the incident light.
  • the amount of light absorbed, scattered, transmitted, fluoresced or phosphoresced can provide an indication of certain physical characteristics of the substance into which an incident light is directed.
  • the amount of light scattered, and the angle of the scattered light relative to the incident light can be used as an index of particle concentration and size.
  • Present instruments that measure light scatter and transmission properties are complex. These instruments typically use a non-solid state illumination source and have poor power efficiencies. Such instruments are useful in nephelometry, turbidity, bacterial suspension assays and biomass determinations.
  • colorimetric examples include ELISA (which is useful in clinical and biological tests) , test tubes coated with antibodies which can be used in clinical and environmental assays, direct determinations (for example pH, where the colorimetric indicator is directly sensitive to the concentration of the analyte oxygen content) , various clinical tests, immunological tests, tests for specific diseases, and classes of colorimetric tests that determine the concentration of individual components within a compound or material.
  • Illumination sources for spectrophotometers typically include either a tungsten element lamp or a rare gas arc lamp, which provide a wide bandwidth output and high intensity.
  • Illumination sources for spectrophotometers typically include either a tungsten element lamp or a rare gas arc lamp, which provide a wide bandwidth output and high intensity.
  • the energy conversion efficiencies of these instruments are low, and they generally are powered by 120 volt ac sources, and dissipate tremendous heat.
  • the tungsten element illumination sources have a relatively short life, are susceptible to damage from vibration, and experience frequency and intensity drift. Circuitry required to stabilize such drift is relatively expensive and complex.
  • the power requirements for most AC powered instruments are over 100 watts.
  • Some instruments of this type incorporate a flashlight-style light bulb, which provides a wide band illumination and thus requires bandpass filtering to remove the wavelengths that would corrupt a particular photometric or colorimetric determination.
  • a diffraction gratings generally require collimated light from a thermally stable optical bench.
  • Optical assemblies are also not flexible in design. Broadband light from tungsten element lamps generally can be filtered with a narrow bandpass dichroic filter. However, dichroic filters shift the bandpass wavelength as the angle of incident light changes. Unstable optical paths, movement of the tungsten element, frequency drift of the lamp, and the deposition of particulate from the heated element onto the inside surface of the glass lamp enclosure all contribute to an unstable system. Such unstable systems have a low resolving power.
  • Photometers and spectrophotometers generally have a fixed design positioned on an optical bench. To change the design, the optical bench must be changed. Often the component values must change to accommodate changes in amplifier gain, and a thermal analysis must be completed to ensure that the new design will accommodate the heat dissipation. The enclosure also must be appropriately modified.
  • the resolving power of a spectrophotometer is, in part, dependent on the instrument's ability to reject stray ambient light. Ambient light from the surrounding environment can add an artifactual offset to the measured light signal. Manufacturers therefore have usually shielded the sample container and the optical path from ambient light. This shielding has been costly and cumbersome.
  • Measuring the collective light intensity and ambient light intensity simultaneously requires two different sets of signal processing components. Each set is subject to its own imprecision and noise errors. Therefore, simple signal subtraction does not necessarily yield a precise indication of the level of the illuminating light intensity.
  • a strategy to overcome this deficiency is to measure many signals from both sets of components over extended periods, thereby integrating the effect of noise. However, such integrating techniques are considered best suited for removing high and very low frequency noise, not for removing the ac component associated with standard ac power sources.
  • Cancellation circuits of the type that may be used to subtract one signal from another signal, are known in the art.
  • Besar, et al. "Simple Fiber Optic Spectrophotometric Cell for pH Determination," J. Biomed. Eng. Vol. II, March 1988, describes a set of shielded photodiodes and two light-emitting diodes (at 565 and 635 nanometers) that may be used for phenol red pH determinations.
  • the anode of one photodiode is connected to the inverting input terminal of an operational amplifier and the cathode of a second photodiode is connected to the operational amplifier's non-inverting input terminal.
  • the opposite photodiode terminals are connected to ground.
  • One photodiode detects light intensities, while the other photodiode normalizes for fluctuations in the light source.
  • An instrumentation amplifier can be substituted for the above operational amplifier.
  • the sensitivity of the isosbestic point to temperature and concentration changes, and the use thereof, are not described in the article. Nor does the article describe operation in a backscatter mode or under ambient light conditions. Imbalances in photodiode response and intrinsic noise in the Besar et al. system leads to large noise values and narrow dynamic range.
  • the present invention fulfills this need.
  • the present invention relates to a device that determines an optical intensity of light emanating from a sample situated under a periodically varying ambient light.
  • the device includes a selectively illuminating light source that can be switched between on and off states that illuminates the sample.
  • a detector detects the optical intensities of the sample a first plurality of times with the effects of the illuminating light source to produce a plurality of collective light intensity measurements, and a second plurality of times without the effects of the light source to produce a plurality of ambient light intensity measurements.
  • a processor quantitatively determines, based upon the multiple collective light intensity measurements and the multiple ambient light intensity measurements, the intensity amount of each of the collective light intensity measurements that results from said illuminating light source.
  • a feature of the present invention involves doing the above detecting in the presence of ambient light.
  • the ambient light has a high frequency component, a 60 Hz component that results from the frequency of the power source, and a low frequency component.
  • the frequencies at which the filters function can be adjusted depending upon the specifics of the ambient light being provided and the optical intensity of the sample.
  • Another feature of the present invention is that the system functions effectively whether the sample is contained in a t-flask, pipette, cuvette, of any other well known type of container that the light can pass through.
  • the present invention may be applied to measure the optical intensity of a sample by itself, a sample with an agent contained in it, a sample with particulates in it, or any other type of sample.
  • the measured intensity can be a result of fluorescence, transmittance, particulate refraction, or other types of optical phenomenon.
  • FIG. 2 is a partial cross-sectional view as taken through section lines 2-2 of FIG. 1, with a container holding a sample positioned above the light intensity measuring device;
  • FIG. 3 is a block diagram of one embodiment of light intensity measuring device of the present invention that may be applied to the FIGS. 1 and 2 light intensity-measuring apparatus;
  • FIG. 6 is a top elevational view illustrating light pathways in an alternative embodiment of the light intensity measuring apparatus of the present invention.
  • FIG. 7 is a block diagram illustrating the light pathways in a final embodiment of light intensity measuring apparatus of the present invention.
  • FIGS. 1 and 2 there is shown a first embodiment of an apparatus 20 for measuring the intensity of light of a particular wavelength emitted from a liquid medium or sample 22.
  • the sample is contained in a container 24, and is exposed to background, ambient light of varying intensity.
  • a pair of light-emitting diodes (LEDs) 26a and 26b and a pair of photodiodes 28a and 28b are located on a printed circuit board 30, which is positioned immediately beneath the container (in this case, a t-flask) .
  • LEDs light-emitting diodes
  • the term "LED" is used even though a laser diode may be substituted therefore, and the term used herein is intended to cover both types of light emitting diodes.
  • the photodiode 28a is positioned between the two LEDs, e.g., 1 to 5 millimeters (mm) away, and the photodiode 28b is spaced further away, e.g., 5 to 25 mm away, such that it receives relatively less light from the sample.
  • mm millimeters
  • the photodiode 28b is spaced further away, e.g., 5 to 25 mm away, such that it receives relatively less light from the sample.
  • any number of LEDs may be used.
  • the apparatus 20 includes a light intensity detection circuit 36, one embodiment of which is depicted in FIG. 3. This circuit measures and stores the intensity of light impinging on the photodiodes 28a and 28b. The measurement made from the photodiode
  • DSP digital signal processor
  • the two photodiodes 28a and 28b are arranged in a parallel, reverse-biased relationship, with one terminal connected to the op-amp's inverting input terminal and the other terminal connected through a resistor 46 to an adjustable voltage reference provided by a first resistor divider 48.
  • the resistor 46 preferably has a resistance of about 4 mega-ohms.
  • a second resistor divider 50 is connected to the op-amp's non ⁇ inverting input terminal, and a second resistor 52 is connected between the op-amp's output and inverting input terminals.
  • the two resistor dividers are adjusted to provide the desired dc voltage levels to the op-amp's two input terminals.
  • the resistances of the photodiodes 28a and 28b generally vary linearly with the intensity of incident light. Thus, if the two photodiodes receive equal amounts of light, no net electrical current will be produced. On the other hand, if one photodiode receives more incident light than does the other, a net positive or negative current will be produced, which the op-amp 38 converts into a voltage output signal of corresponding amplitude.
  • the ADC 40 converts this voltage into a corresponding sequence of digital words, at a prescribed sample rate, and these words are supplied on lines to the microcontroller 44/DSP 42, for further processing. Operation of the LEDs 26a and 26b and the ADC are controlled by the microcontroller 44/DSP 42.
  • an LED output compensation circuit (not shown) is included.
  • a commercially available constant current source is connected from a voltage supply to a node, and provides a constant current to the node.
  • the LED and a parallel-connected resistor are connected from the node to ground in parallel.
  • the intensity of the light emitted by the LED varies as a function of both internal temperature and external temperature. As the intensity of the emitted light changes, the internal resistance of the LED also changes.
  • the parallel-connected resistor which preferably has a resistance between 10 - 5,000 ohms, functions as a current shunt.
  • the microcontroller 44/DSP 42 controls the LEDs 26a and 26b such that they receive pulses of electrical current, at a frequency of 2000 Hz and a duty cycle of 10%.
  • the current preferably pulses between 2 and 200 milliamps. Other frequencies and duty cycles alternatively can be used.
  • the LEDs preferably emit light having wavelengths between 420 and 3900 nm.
  • the more remote photodetector 28b typically receives a lower intensity of illuminating light from the sample than does the photodetector 28a. Since some illuminating light that emanates from the LEDs 26a, 26b is received by both photodetectors, the calculations associated with subtracting the ambient light appear to become very complex.
  • calibrations can be used to compare the resultant output light intensities of an unknown sample with one or more resultant output light intensities of samples that have a known optical characteristic. Such known optical characteristics may result from, for example, using a control sample having a known pH. Improved accuracy can be obtained by adding additional calibration steps with a reference sample having a known pH.
  • Other calibration techniques also can be used, for example, color changes present in a base tetramer and an acid tetramer, which can be ratioed to obtain a value related to pH.
  • FIG. 4 depicts an alternative embodiment of a light intensity detection circuit 56 for measuring the intensity of light emitted from the sample 22 in the presence of ambient light and light produced selectively from a LED 61.
  • This embodiment incorporates only digital circuitry.
  • the FIG. 4 circuit differs from the circuit 36 of FIG. 3 in that light emitted from the sample is not detected by two photodiodes, as described above, but rather by a single light-to-frequency converter 58, which produces digital signals whose frequencies vary substantially directly with the intensity of impinging light.
  • the light-to-frequency converter includes a photodetector and a voltage-controlled oscillator integrated to the same substrate.
  • One suitable light-to-frequency converter is available from Texas Instruments, under the part number TSL230.
  • the DSP portion 60 of the microcontroller 44 of FIG. 4 is configured to count the number of pulses produced by the light-to- frequency converter 58 for prescribed time periods, preferably over a period that lasts for an integral multiple of 1/60 of a second. This provides a measure of the intensity of light impinging on the converter.
  • the light intensity detection circuit 56 makes optical measurements with the LED 61 biased OFF, to measure the contribution of ambient light. Also the circuit makes optical measurements with the LED biased ON, to measure the combined effects of ambient light and light from the LED.
  • This section describes signal processing techniques of the DSP portion 42 (FIG. 3) or the DSP portion 60 (FIG. 4) of the microcontroller. Both signal processing techniques can be used effectively to filter out the effects of ambient light, while accurately measuring the illuminating light produced by the LED 26a, 26b (FIG. 3), or 61 (FIG. 4). Ambient light typically varies substantially with time, with that variation including a low- frequency (or dc) component, an ac power line component (60 Hz in the U.S.), and a high-frequency component.
  • the preferred embodiment of the present invention uses three distinct DSP filtering techniques. Each filtering technique removes the effects of a separate component of ambient light.
  • the signal can be transferred to a computer or a variety of electronic media to be stored and monitored, and to provide further system capabilities.
  • the first filtering technique implemented by the DSP 42 or 60 which is akin to high-pass filtering, involves modulating and demodulating the signal produced by the LED 61 synchronously with the sampling by the light-to-frequency converters in the FIG. 4 embodiment, and measuring the light intensity using the photodetectors of FIG. 3, and the light-to-frequency converters of FIG. 4, at a rate considerably higher than the periodic rate of the ac current.
  • a first set of intensity measurements (in both the FIGS. 3 and 4 embodiments) are made while the LED is OFF, and a second set of intensity measurements are made while the LED is ON.
  • This cycling of the LEDs on and off, and the synchronized measuring of the light intensity by the photodiodes or the light- to-frequency converter preferably occurs at a very high rate such as over a hundred times in a fraction of l/60th of a second.
  • the second filtering technique which is akin to notch filtering, removes abnormalities that are due to ambient light variations occurring at the frequency of ac power, and at harmonics of that ac power frequency.
  • This technique involves sampling the signal produced by the op-amp 38 (FIG. 3) , or the light-to- frequency converter 58 (FIG. 4), over a l/60th second sampling period, or a multiple thereof (such as l/60th, 2/60th, 3/60th, . . . , 60/60th of a second, up to and including several seconds)
  • the l/60th second sampling periods provide a 60 Hz notch filter effect.
  • the third filtering technique which is akin to lowpass filtering, removes noise occurring at relatively high frequencies. It is accomplished by sampling the signal often within a relatively short duration, e.g., 128 times over a period of considerably less than 1/60 second, and then averaging the many samples. Momentary aberrations resulting from high-frequency noise components, thereby, are removed. Alternatively, this averaging can be substituted by digital lowpass filtering.
  • the preferred embodiments of the present invention combines the three filtering techniques described in the previous three paragraphs.
  • the unique combination of these three filtering techniques effectively filters out the effects of ambient light from a signal representing the combined light intensities of ambient light and light resulting from an illuminating light source.
  • This unique combination led to the discovery that the pH concentration and temperature errors could be determined and controlled in phenol red colorimeter determinators that are under near the isosbestic point of phenol red (approximately 460 to 480 nm) , as now described.
  • FIGS. 1 to 4 show that the apparatus can measure a low level of illuminating light emanating from a medium or sample 22, which is exposed to a relatively high level of ambient light.
  • pH An example of a property of certain samples, for which the illuminating light intensity of the samples provides important information, is pH.
  • the pH of a sample may be tested by adding an agent (e.g., phenol red) that alters the color of the sample, and by then detecting the illuminating intensity emitted from the sample at prescribed wavelengths.
  • Phenol red is applied at a preferred concentration of 5 ng/ml to 20 mg/ml, as is known in the medical testing field. Phenol red changes color based upon temperature and concentration, and it is only slightly influenced by the samples' pH at wavelengths of 470 nm.
  • phenol red is a unique reference since it acts as a control for concentration and temperature changes, and it can be used to normalize colorimetric pH determinations at various concentrations and temperatures.
  • the color intensity is sensitive to temperature or concentration changes, as described in Besar, S.S.A. et al., "Simple Fiber Optic Spectrophotometric Cell for pH Determination," J. Biomed. Eng. Vol. 11, March 1988. Consequently, these changes cannot be normalized.
  • an averaged 630 nm was divided by an averaged 470 nm absorption signal reference to correct for temperature and concentration variations. The result is related to the samples' pH.
  • the apparatus 20 has particular utility for the sample 22 contained in the t-flask 24 used for the cell culture.
  • the LED 26a emits light at a wavelength of 470 nm
  • the LED 26b emits light at a wavelength of 630 nm.
  • the 470 nm LED is used as a reference.
  • Light from LEDs 26a, 26b is directed at (and through) a bottom wall 24 of the t-flask, into the liquid sample 22.
  • a 25 ml t-flask having 2 to 25 ml of cell culture sample containing a prescribed amount of phenol red has been found suitable.
  • the LED light from LEDs 26a, 26b follows respective paths 66a, 66b through the sample, reflected off a sample/air interface 32 and follows respective paths 70a, 70b back through the sample.
  • the light following the paths 70a and 70b is backscattered such that the light intensity of each path is detected by the light intensity detection circuit 36.
  • the light intensities received by the light intensity measuring device are processed as described in the signal processing section, to obtain the signal intensity under ambient overhead lighting conditions.
  • each LED 26a, 26b is processed in the same manner.
  • the light generated by LED 26a is used to normalize for temperature and concentration variations of the agent in the sample.
  • the light from the LED 26b is absorbed by the sample containing phenol red (wavelengths from 500 nm to 590 nm will work) in related to the resultant intensity of the sample.
  • the light output of a 565 nm LED 74 is directed along at least one light path 76 through a pipette tip 78 into a light intensity detection circuit 80 (of the type illustrated either in FIGS. 3 or 4) .
  • a 200 ⁇ l pipette tip (as produced by SigmaTM, St. Louis, MO) is suitable.
  • the pipette tip 78 containing water is interposed in the light path between the LED and the light intensity measuring device to act as a control.
  • the number of pulses counted, after signal processing, is proportional to a 100% light transmission.
  • a second pipette tip of similar size and design is filled with the sample (assume, for example, a 2% red dye) and interposed as before in the light path 76.
  • the red dye absorbs green light of 565 nm, and therefore diminishes the green light received by the light intensity measuring device.
  • the number of output pulses counted from the light intensity measuring device is less than the number counted by the 100% transmission reference. Diminution of the light received by the detector is related to the red dye concentration as governed by Lambert's Law.
  • light from a LED 80 (preferably 459 nm, though various wavelengths will work) is directed along light path 82 toward a sample 84 contained in a container 86 to measure fluorescence of the sample.
  • the light intensity detection circuit 88 (of the type illustrated in FIGS. 3 or 4) is oriented at a right angle to the light path 82 facing the sample. Some light that encounters the sample causes the sample to emanate light based upon fluorescent principles.
  • the fluorescent light follows light paths 90a, 90b, 90c, 90d, and other non-shown light paths spaced about the periphery of the container.
  • the wavelength of the light following paths 90a, 90b, and 90c usually differ from the wavelength of the light following path 82.
  • An absorbent filter 92 is interposed between the light intensity detection circuit 88 and the container 86.
  • the filter filters out the light at the incident wavelengths, while permitting light at the fluorescent wavelengths to pass.
  • the absorbent filter and the light intensity measuring device are oriented 90 degrees to the light path 82, to reduce the flux of excitation photons on the filter spatially.
  • the absorbent filter may be a dichroic filter or an absorption filter.
  • the absorbent filter is selected to allow light that is of the wavelength range(s) of the fluorescent emissions to pass, while absorbing the light that is of the wavelength range(s) produced directly from the LED 80.
  • the only light that the light intensity measuring device receives is the ambient light and the filtered fluorescent light. This filter wavelength selection is generally known by those skilled in the art.
  • the light received by the light intensity measuring device is translated into an output signal, which is processed by the DSP portion of the microcontroller as previously described.
  • the level of fluorescent emissions relates to the number of pulses. Thus, most of the LED light does not reach the filter, but the spherically radiated fluorescent excitation light will reach the filter and pass through to the detector.
  • multiple photodetectors are placed around a pipette such that the detector is not in the direct light path of the LED.
  • Each detector faces toward the sample, and has an absorbent optical filter (similar to 92 above) interposed between the detector and the pipette tip.
  • Each filter has different band pass properties so that each detector views the intensity of the light provided by a different color fluorescent light.
  • the intensity of light scattered by particles in the sample, and thereupon received by the light intensity detection circuit 94c is related to the particulate size and concentration of the particulate matter, and can be compared with the other light intensities detected by the light intensity detection circuits 94a, 94b.
  • an estimate of the concentration and size of particles in solution can be obtained. Scatter and absorbance information is useful in calculating turbidity, colorimetric densities, numbers of bacteria in solution, concentration of pollutes, biomass, and concentration of molecules or compounds.
  • Certain embodiments of the present invention may be applied to a variety of applications.
  • a commercially available TMBTM is used as the chromogen.
  • a sample reacts in a cuvette using horseradish peroxidase as the catalyst.
  • the light intensity measuring devices shown herein can be used in other, non-pH environmental assays as well.
  • an antibody to a known toxin e. g. , PCB
  • a solution containing an unknown amount of PCB is then added to the test tube.
  • An enzyme labeled antibody is then added to the test tube and allowed to react. After the reaction, the non-bound antibody is removed and a chromogen added.
  • the chromogen produces a color change that is related to the PCB concentration. Colorimetric determinations (which may use the embodiments illustrated in FIGS.
  • FIG. 7 embodiment can be taken at a narrow angle ( ⁇ 90°) and a large angle (approximately 90°), to be used for size, concentration, bacterial suspensions, turbidity and biomass determinations.
  • Bacterial suspensions could be used for antibiotic and toxicity testing. Bacterial suspensions of known concentration can be suspended in a solution and analyzed for 630 nm light scatter.
  • a toxic analyte can be added to the bacterial suspension.
  • the solution clears and a lower light intensity is read by the photodiode or the light-to-frequency converter which is positioned perpendicular to the LED normal.
  • the reduction of the light scatter is related to the concentration of the toxin.
  • the bacteria can be preselected for intolerance to a specific substance or genetically altered to become intolerant to a specific substance.
  • Toxins include a variety of organic and inorganic materials.
  • the toxins can include toxic bacterial or cells, inorganic pollutants, antibiotics, peptides, proteins, hydrophobic materials and temperature sensitive materials.
  • the present invention can be used to adjust inoculum density against a McFarland Standard.
  • the present invention can be calibrated with McFarland standards.
  • Light scatter from an unknown concentration of bacteria can be compared to controls that have known bacterial concentrations.
  • the bacterial suspension is then adjusted, by adding additional bacteria or diluting the solution, to achieve the desired light scatter.
  • an apparatus was constructed as illustrated in FIG. 7, with a light intensity detection circuit located at 90° to the LED normal and a second light intensity detection circuit juxtaposed to the LED normal.
  • Commercially available nephelometry standards or calibration standards for turbidity measurements are placed in round test tubes and placed into the apparatus. Light scatter is measured and displayed on the LCD display.
  • commercially available McFarland standards are obtained to establish bacterial suspension calibration. The standards are placed in round test tubes and scatter measured. Results are displayed on the LCD display. The resultant resolution is sufficient to resolve 0.1 McFarland units in a range of 0.1 to 5.0 Alternatively, the light collection from photodetectors (FIG. 3) or light-to-frequency converters (FIG.
  • the wavelengths tested were 590 nm, 630 nm, and 660 nm, one or more other wavelengths could be used including 625 nm.
  • the synchronous modulation and demodulation of the LED with each light intensity detection circuit is controlled by the microcontroller.
  • the power to the LED is turned on/off by the microcontroller to synchronously modulate and demodulate the signal.
  • Light from LED is directed at, and controlled by, the light intensity detection circuit to determine how much light is absorbed, scattered, transmitted, or fluoresced by the contents of the sample container. Sample containers are held in place by the sample guide positioned within the sample insertion hole.
  • the microcontroller turns the power to the light intensity measuring circuits on when the signal is collected and off when the light intensity measuring circuit not required, thereby conserving battery power.
  • the light-to-frequency converters provide a 50% duty cycle at a frequency that is proportional to the incident light.
  • the microcontroller collects the output signal from the light intensity detection circuit.
  • the output of the light intensity detection circuit has a frequency that proportionally ranges to the input from 0 Hz to 500,000 Hz.
  • the signal is acquired when the microcontroller counts the number of pulses generated during a fixed time interval. The number of pulses counted is related to the wavelengths of the incident light, and the resultant light intensity emitted from the sample.
  • AlamarBlue also has an excitation/emission spectra change as a function of cell viability in the sample to somewhere between the wavelenghts of 560 nm and 590 nm, respectively.
  • Another example of applying cells to the present apparatus is to detect sperm viability using two color fluorescence by replacing the sample with sperm mixed with live/dead FertLightTM (produced by Molecular Probes, Inc., OR).
  • a variety of containers can contain the sample for any of the above embodiments. These containers include, but not limited to, cuvettes, pipettes, t-flasks, test tubes, and virtually any container that light can pass through such that the optical characteristics of the sample can be measured.
  • different configurations may be used to measure light emanating from sample under different mechanisms> such as fluorescence colorimetric measurements, diffraction, transmittance, and reflectance.
  • Colorimetric and fluorescent measurements associated with special signal processing methods, can be used to detect pH of the sample under ambient light optically, without external temperature regulation.
  • a container having static fluid can be replaced by a tube through which fluid flows. Colorimetric, fluorescent, diffraction, transmittance and reflectance changes over time can be monitored.
  • Preferred embodiments of the present invention resolve at least to 0.5 parts in 1000, have a dynamic range beyond 3.0 optical densities, have a 1% variation from linearity over the entire range, and have a battery life of 2 years that is sufficient for thousands of tests.
  • the produced apparatus is solid state, is pocket-calculator in size, does not have an optical assembly, displays a 4 digit result, and does not require ambient light shielding. These characteristics make the present system easier to use accurately than the prior art systems.
  • the LED illumination source is preferably solid state, is efficient, is narrow band, consumes low power because of the intrinsic high efficiency, and does not require a warm-up period.
  • the preferred embodiment uses a microcontroller that allows the apparatus to always be at least partially on and to turn the desired components on or off as needed.
  • the present invention uses a LED on a standard printed circuit board without an optical bench, collimating optics, focusing optics or diffraction gratings.

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  • Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • General Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Sustainable Development (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Un dispositif (20) détermine l'intensité optique d'une lumière provenant d'un échantillon (22) situé sous une lumière ambiante (34) variant de manière périodique. Le dispositif (20) comprend une source de lumière (26a, 26b) à éclairage sélectif qui peut être commutée entre un état d'éclairage de l'échantillon (22) et un état éteint. Un détecteur (36) détecte les intensités optiques de l'échantillon (22) pendant une première pluralité de durées lorsque la source de lumière d'éclairage (26a, 26b) est allumée, pour produire une pluralité de mesures collectives de l'intensité de la lumière, et une seconde pluralité de durées sans les effets de la source de lumière (26a, 26b) pour produire une pluralité de mesures d'intensité de la lumière ambiante. Un processeur détermine quantitativement, à partir des multiples mesures collectives de l'intensité de la lumière et des multiples mesures de l'intensité de la lumière ambiante, la quantité d'intensité de chacune des mesures collectives de l'intensité lumineuse collective résultant de ladite source d'éclairage (26a, 26b).
PCT/US1996/013523 1994-11-01 1996-08-22 Procede et appareil de determination des caracteristiques d'un echantillon en presence de lumiere ambiante Ceased WO1997008523A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU68536/96A AU738290B2 (en) 1995-08-22 1996-08-22 Method and apparatus for determining characteristics of a sample in the presence of ambient light
JP9510404A JPH11511557A (ja) 1995-08-22 1996-08-22 環境光の下での標本の特性を決定する方法と装置
EP96928966A EP0866953A4 (fr) 1995-08-22 1996-08-22 Procede et appareil de determination des caracteristiques d'un echantillon en presence de lumiere ambiante

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US33272894A 1994-11-01 1994-11-01
US263595P 1995-08-22 1995-08-22
US60/002,635 1995-08-22

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WO1997008523A1 true WO1997008523A1 (fr) 1997-03-06

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JP (1) JPH11511557A (fr)
AU (1) AU738290B2 (fr)
CA (1) CA2229458A1 (fr)
WO (1) WO1997008523A1 (fr)

Cited By (17)

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WO1999060380A1 (fr) * 1998-05-19 1999-11-25 Cepheid Systeme de detection optique a plusieurs voies
WO2001063253A1 (fr) * 2000-02-24 2001-08-30 Eppendorf Ag Systeme de mesure optique
EP1203944A1 (fr) * 2000-11-06 2002-05-08 Hofstetter, Alfons, Prof.Dr.med. Dispositif d'identification de cellules turmorales et procédé d'identification de cellules turmorales
EP1035409A3 (fr) * 1999-02-12 2002-08-07 Fuji Photo Film Co., Ltd. Procédé et dispositif de mesure de la lumière émise par un échantillon éclairé permettant l'élimination de l'influence de lumière de fond
ES2171138A1 (es) * 2000-12-11 2002-08-16 Univ Santiago Compostela Determinacion de biomasa y actividad biologica de organismos fototroficos en medio liquido y sobre materiales solidos a partir de la medida del color.
WO2003008948A1 (fr) * 2001-07-17 2003-01-30 Consejo Superior De Investigaciones Cientificas Equipement et methode en ligne de detection, de determination de l'evolution et de quantification d'une biomasse microbienne et autres substances absorbantes sur le spectre de lumiere dans la mise au point de processus biotechnologiques
US6658138B1 (en) * 2000-08-16 2003-12-02 Ncr Corporation Produce texture data collecting apparatus and method
EP1750106A1 (fr) 2005-08-04 2007-02-07 Hach Lange GmbH Spectrophotomètre pour des mesures avec une cellule de mesure ouverte à la lumière ambiente
EP2306233A3 (fr) * 2004-11-04 2011-07-20 Life Technologies Corporation Systèmes et procédés de balayage optique a compensation thermique
EP2462426A1 (fr) * 2009-08-05 2012-06-13 Georg Fritzmeier GmbH + Co. KG Dispositif de mesure pour la détermination d'une valeur d'indice de végétation de plantes
WO2016074041A1 (fr) 2014-11-14 2016-05-19 Nplex Pty Ltd Detecteur diagnostique in vitro portatif et appareil
EP3234526A1 (fr) * 2014-12-18 2017-10-25 Merck Patent GmbH Spectrophotomètre et procédé d'exécution d'une mesure spectrophotométrique
US10436699B2 (en) 2014-10-20 2019-10-08 Nihon Kohden Corporation Analyzing system and analyzing apparatus
CN110869749A (zh) * 2017-08-29 2020-03-06 松下知识产权经营株式会社 光观测装置
US20200323700A1 (en) * 2019-04-12 2020-10-15 Verily Life Sciences Llc Determining diaper loading using color detection or activity state
CN114030640A (zh) * 2021-11-05 2022-02-11 中国航空无线电电子研究所 用于对飞机驾驶舱光环境进行评估的方法
US11607143B2 (en) 2019-04-12 2023-03-21 Verily Life Sciences Llc Sensing physiological parameters through an article

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JP3727494B2 (ja) * 1999-08-26 2005-12-14 富士写真フイルム株式会社 光計測方法および装置
JP2018116060A (ja) * 2018-02-09 2018-07-26 日本光電工業株式会社 画像サイトメータ

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Cited By (29)

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AU746069B2 (en) * 1998-05-19 2002-04-11 Cepheid Multi-channel optical detection system
JP2002515602A (ja) * 1998-05-19 2002-05-28 シーフィード マルチチャンネル光検出装置
WO1999060380A1 (fr) * 1998-05-19 1999-11-25 Cepheid Systeme de detection optique a plusieurs voies
EP1035409A3 (fr) * 1999-02-12 2002-08-07 Fuji Photo Film Co., Ltd. Procédé et dispositif de mesure de la lumière émise par un échantillon éclairé permettant l'élimination de l'influence de lumière de fond
US6597439B1 (en) 1999-02-12 2003-07-22 Fuji Photo Film Co., Ltd. Method and apparatus for measurement of light from illuminated specimen eliminating influence of background light
US9285318B2 (en) 1999-05-17 2016-03-15 Applied Biosystems, Llc Optical instrument including excitation source
WO2001063253A1 (fr) * 2000-02-24 2001-08-30 Eppendorf Ag Systeme de mesure optique
US6658138B1 (en) * 2000-08-16 2003-12-02 Ncr Corporation Produce texture data collecting apparatus and method
EP1203944A1 (fr) * 2000-11-06 2002-05-08 Hofstetter, Alfons, Prof.Dr.med. Dispositif d'identification de cellules turmorales et procédé d'identification de cellules turmorales
ES2171138A1 (es) * 2000-12-11 2002-08-16 Univ Santiago Compostela Determinacion de biomasa y actividad biologica de organismos fototroficos en medio liquido y sobre materiales solidos a partir de la medida del color.
WO2003008948A1 (fr) * 2001-07-17 2003-01-30 Consejo Superior De Investigaciones Cientificas Equipement et methode en ligne de detection, de determination de l'evolution et de quantification d'une biomasse microbienne et autres substances absorbantes sur le spectre de lumiere dans la mise au point de processus biotechnologiques
ES2185496A1 (es) * 2001-07-17 2003-04-16 Univ Valencia Politecnica Equipo y metodo en linea para la deteccion, determinacion de la evolucion y cuantificacion de biomasa microbiana y otras sustancias que absorben a lo largo del espectro de luz durante el desarrollo de procesos biotecnologicos
EP2306233A3 (fr) * 2004-11-04 2011-07-20 Life Technologies Corporation Systèmes et procédés de balayage optique a compensation thermique
EP1750106A1 (fr) 2005-08-04 2007-02-07 Hach Lange GmbH Spectrophotomètre pour des mesures avec une cellule de mesure ouverte à la lumière ambiente
EP2462426A1 (fr) * 2009-08-05 2012-06-13 Georg Fritzmeier GmbH + Co. KG Dispositif de mesure pour la détermination d'une valeur d'indice de végétation de plantes
US10436699B2 (en) 2014-10-20 2019-10-08 Nihon Kohden Corporation Analyzing system and analyzing apparatus
US11112362B2 (en) 2014-11-14 2021-09-07 Lumos Diagnostics IP Pty Ltd Portable in-vitro diagnostic detector and apparatus
WO2016074041A1 (fr) 2014-11-14 2016-05-19 Nplex Pty Ltd Detecteur diagnostique in vitro portatif et appareil
EP3218696A4 (fr) * 2014-11-14 2018-05-30 Nplex Pty Ltd Detecteur diagnostique in vitro portatif et appareil
EP3234526A1 (fr) * 2014-12-18 2017-10-25 Merck Patent GmbH Spectrophotomètre et procédé d'exécution d'une mesure spectrophotométrique
CN110869749A (zh) * 2017-08-29 2020-03-06 松下知识产权经营株式会社 光观测装置
US20200323700A1 (en) * 2019-04-12 2020-10-15 Verily Life Sciences Llc Determining diaper loading using color detection or activity state
US11607143B2 (en) 2019-04-12 2023-03-21 Verily Life Sciences Llc Sensing physiological parameters through an article
US11679036B2 (en) * 2019-04-12 2023-06-20 Verily Life Sciences Llc Determining diaper loading using color detection or activity state
US20230248584A1 (en) * 2019-04-12 2023-08-10 Verily Life Sciences Llc Determining diaper loading using color detection or activity state
US12369804B2 (en) 2019-04-12 2025-07-29 Verily Life Sciences Llc Sensing physiological parameters through an article
US12458543B2 (en) * 2019-04-12 2025-11-04 Verily Life Sciences Llc Determining diaper loading using color detection or activity state
CN114030640A (zh) * 2021-11-05 2022-02-11 中国航空无线电电子研究所 用于对飞机驾驶舱光环境进行评估的方法
CN114030640B (zh) * 2021-11-05 2023-10-27 中国航空无线电电子研究所 用于对飞机驾驶舱光环境进行评估的方法

Also Published As

Publication number Publication date
EP0866953A4 (fr) 2000-05-24
EP0866953A1 (fr) 1998-09-30
CA2229458A1 (fr) 1997-03-06
AU6853696A (en) 1997-03-19
JPH11511557A (ja) 1999-10-05
AU738290B2 (en) 2001-09-13

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