CN103323410A - A device and a method based on a liquid-crystal filtering device for microscopic spectral imaging - Google Patents

Abstract

The invention discloses a microscopic spectrum imaging device and method based on a liquid crystal filter device. The device includes a light source, a lens barrel, a sample stage, a CCD for fluorescence imaging, a bracket, a computer, a microscopic objective lens, and is used for magnifying The LCTF and imaging lens assembly for filtering the image formed after displaying, the light source is set at the corresponding position according to the incident mode, the lens barrel is fixed on the bracket, the sample stage is set on the bracket base, and the CCD is set at one end of the lens barrel and connected to the computer , CCD, lens barrel, imaging lens assembly, LCTF, and microscope objective lens are located on the same optical axis, the sample is placed on the sample stage directly below the microscope objective lens, and the LCTF is connected to the computer. The method is to enlarge the sample through the microscope objective lens, and obtain the imaging of the enlarged part of the sample at each wavelength in a certain waveband by controlling the scanning range and scanning step of the liquid crystal filter device. The invention can quickly and accurately obtain high-quality microscopic imaging, continuously adjustable magnification and wide applicability.

Description

A microspectral imaging device and method based on a liquid crystal filter device

technical field

The invention relates to the technical field of spectral analysis and microscopic imaging, in particular to a microspectral imaging device and method based on a liquid crystal filter device.

Background technique

The nature of the absorption, emission or scattering of light by matter is the interaction between light and matter. Molecular spectra can be obtained by studying this action process, that is, the change of light intensity with frequency and drawing the corresponding curve. According to the wavelength range and action form of light radiation, molecular spectroscopy can be divided into ultraviolet-visible spectroscopy, fluorescence spectroscopy, Raman spectroscopy, two-photon fluorescence spectroscopy, infrared spectroscopy, etc. Different spectra can provide researchers with different information inside the molecule. Spectral detection technology has the characteristics of high sensitivity and good specificity, and has become one of the commonly used methods in modern detection technology. It is widely used in pharmaceutical analysis, food hygiene, environmental detection, industry and agriculture, geological exploration, judicial criminal investigation and other fields.

Such as the use of spectroscopy to analyze the components of traditional Chinese medicine [Zhao Jing, Pang Qichang, Ma Ji, Liu Chuanming, Wang Lin, Cui Daijun, Meng Qingxia, Spectral imaging analysis technology of Chinese medicine Coptidis Rhizome and Cortex Phellodendri mixed powder. Acta Optics Sinica, 2010, 3(11): 3259-3263] . Another example is that as early as 2003, Australia's Claude Roux et al. used a liquid crystal tunable wavelength filter (LCTF) spectral imaging device to conduct a preliminary experimental study on the inspection of fingerprint lines [Exline DL, Wallace C, Roux C, et al. Forensic applications of chemical imaging: latent fingerprint detection using visible absorption and luminescence [J], Forensic science. 2003, 48 (5): 1-7]. With the advancement of photoelectric technology, such as light source, imaging equipment, detection equipment, etc., the spectral analysis technology has penetrated into the field of microscopic imaging and is highly integrated with biology and medicine. For example, the method of spectral analysis is used to detect the apoptosis process of cells [National Human GenomeResearch Institute.NHGRI seeks next generation of sequencing technologies.http://www.genome.gov/12513210.2009,11,4], and FT -IR Microscopic Spectroscopy for Cluster Analysis of Colorectal Cancer 2004,1688:176-186], there are also related reports on the observation of arterial microstructure by second harmonic wave and two-photon fluorescence [Aikaterini Zoumi, Xiao Lu, Ghassan S.Kassab, and Bruce J.Tromberg. Imaging Coronary Artery Microstructure Using Second-Harmonic and Two-Photon Fluorescence Microscopy[J], Biophysical Journal.2004,87:2778–2786]. The combination of spectral analysis and microscopic imaging has developed a series of new detection systems, expanded the research field of biomedicine and deepened the research depth of many microscopic tissues.

At present, in order to obtain spectral images under microscopic conditions, filters, prisms and grating spectrometers (monochromators) are generally used for filtering, so as to obtain images at certain or certain wavelengths on the imaging CCD. For example, in the multi-spectral camera produced by Quest innovations, optical prism light splitting and filter wheel filtering are used, which are widely used in remote sensing and microscopic imaging. Using filters or prisms to filter or split light, the spectral images obtained are relatively scattered, and only certain imaging under certain specific spectra can be obtained, but its complete and wide-ranging spectral characteristics cannot be obtained. Various hyperspectral imagers launched by Specim use grating spectrometers for filtering, which have a long wavelength range (according to different types, the wavelength span of the filter is generally above 500nm) and high precision (spectral resolution below 10nm) , but using a hyperspectral imager combined with a grating spectrometer + CCD, each imaging is only a line (one-dimensional). To obtain a two-dimensional image of the entire sample, the sample needs to be pushed and broomed and photographed, and finally the images are stitched together. . This method not only needs to scan the wavelength, but also needs to push and broom the sample in the process of acquiring the spectral image. The imaging time is long, and the image stitching will also generate additional noise, so it also has its shortcomings. In order to quickly and accurately obtain two-dimensional spectral images and corresponding spectral curves of samples, a liquid crystal filter device (LCTF) + CCD mode has been developed. There are not many reports on the acquisition of spectral imaging using the liquid crystal filter device (LCTF) + CCD mode, mainly focusing on the acquisition of spectral images of macroscopic objects (Weilin Wang, Changying Li, Ernest W. Tollner, Glen C. Rains, Ronald D. Gitaitis, A liquid crystal tunable filter based shortwave infrared spectral imaging system: Design and integration [J], Computers and Electronics in Agriculture. 2002, 80: 126-134), also use this method to measure blood pH value and dynamic response in blood vessels [Xiaoli Sun, Yaru Wang, Shangbin Chen, Weihua Luo, Pengcheng Li, Qingming Luo, Simultaneous monitoring of intracellular pH changes and hemodynamic responding cortical spreading depression by fluorescence-corrected multimodal optical imaging1,5[J], 7 -884]. However, there is still a lack of a complete set of LCTF+CCD systems for microscopic systems. The main problems are as follows: First, for the traditional micro-optical system, many optical components need to be added in the middle of the spectral micro-system, and it is difficult to obtain spectral imaging while ensuring the quality of the microscopic image; secondly, due to the In microscopic imaging, the imaging area is very small, that is, the light that can enter the CCD is very small. When the light energy is evenly distributed on the CCD surface, the energy density on the surface is very small. Two-photon fluorescence spectral signals, Raman spectral signals, etc., are quite difficult.

Therefore, the development of a method and related equipment system that can quickly and accurately obtain spectral images under a microscope system has great scientific and application significance, and provides strong support for further expanding the application of spectral analysis in biomedicine.

Contents of the invention

The main purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, to provide a microspectral imaging device based on liquid crystal filter devices, which can quickly and accurately obtain high-quality microscopic imaging, and only need The scanning spectrum does not need to scan the sample, and has wide applicability.

The present invention also provides a microspectral imaging method based on the above microspectral imaging device, the method does not need to perform push-broom and image splicing, presents a two-dimensional image in real time, and can directly take points of interest in the image And obtain the spectral curve of this point.

The purpose of the present invention is achieved through the following technical solutions: a microspectral imaging device based on liquid crystal filter devices, including a light source, lens barrel, sample stage, CCD for fluorescence imaging, a support and a computer, and the light source is arranged in a corresponding position according to the different modes of incidence position, the lens barrel is fixed on the bracket, the sample stage is set on the base of the bracket, and the CCD for fluorescence imaging is set at one end of the lens barrel and connected to the computer. The liquid crystal filter (Liquid Crystal tunable filter, LCTF), imaging lens assembly for filtering the image, CCD for fluorescence imaging, lens barrel, imaging lens assembly, liquid crystal filter, and microscope objective lens are located on the same optical axis, and the sample to be tested is placed On the sample stage directly under the microscope objective lens, the liquid crystal filter device is connected with the computer.

As a preferred solution, the light source is a transmission light source, that is, the light source is arranged under the sample.

As another preferred solution, the light source is a coaxial light source, and the coaxial light source incident system consists of three parts: a coaxial light input hole, a 45-degree total reflection mirror, and a microscopic objective lens. The coaxial light input hole is arranged on the liquid crystal filter device , On the side wall perpendicular to the axis of the optical axis between the microscope objective lenses, a 45-degree total reflection mirror is set in the optical path, and the 45-degree total reflection mirror maintains a 45-degree angle with the optical axis, and the light source enters from the coaxial light input hole. It is reflected by the 45-degree total reflection mirror to the microscope objective lens and irradiated to the sample after being focused, in which the 45-degree total reflection mirror fully reflects infrared light and visible light.

Preferably, the position of the imaging lens assembly in the lens barrel is adjustable. Therefore, by changing its position in the lens barrel, the imaging size of the sample in the CCD for fluorescence imaging can be changed, that is, its magnification can be changed, so that the whole system can realize continuous magnification of the sample.

Preferably, an LCTF with a filter band in the infrared is used for infrared spectrum detection, and an LCTF with a filter band in visible light is used for fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum, and absorption spectrum detection.

Furthermore, the device also includes an ultraviolet filter to prevent the ultraviolet light used to excite fluorescence from entering the liquid crystal filter device. The mirror and the microscope objective are on the same optical axis. So as to avoid ultraviolet light from damaging the liquid crystal filter device.

Preferably, there is a position-adjustable hook on the bracket, and the lens barrel is fixed on the hook by fixing screws. At the same time, a Z-axis fine-tuning knob for adjusting the height of the hook in the vertical direction is also provided at the connection between the hook and the bracket. . The position of the hook on the bracket can be roughly adjusted according to the distance from the objective lens to the sample, and then fine-tuned by the Z-axis fine-tuning knob.

Preferably, the support base is provided with an X-axis fine-tuning knob and a Y-axis fine-tuning knob for adjusting the horizontal position of the sample stage on the support base. Therefore, the enlarged position of the sample can be adjusted through the two fine-tuning knobs without moving the sample stage.

Preferably, the CCD for fluorescence imaging is an infrared CCD for measuring infrared spectrum, or a visible-infrared CCD for measuring fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum, or absorption spectrum.

Preferably, the CCD for fluorescence imaging adopts a cooling method without mechanical vibration. Because the CCD in the present invention requires high sensitivity and the ability to detect weak light, if air-cooled and water-cooled cooling methods with mechanical vibrations are used, the CCD receiving surface array will be affected by mechanical vibrations, causing the imaging lens assembly to be imaged on its surface. Not clear, seriously affecting the image quality.

Furthermore, the CCD for fluorescence imaging adopts a cooling method of liquid nitrogen refrigeration.

Preferably, the microscopic objective lens is a microscopic objective lens of an achromatic plan infinity system. It can be a microscope objective lens of various magnifications. More accurate images can be obtained by using an achromatic microscope objective lens. The infinity system is used to facilitate the addition of various optical elements behind the microscope objective lens, and the restriction on the length of the lens barrel is removed, so that different LCTFs can be replaced at any time , different imaging lens assemblies and different CCDs.

A microscopic spectral imaging method based on the above microscopic spectral imaging device, the sample is enlarged through a microscopic objective lens, and the enlarged part of the sample at each wavelength in a certain band is obtained by controlling the scanning range and scanning step of the liquid crystal filter device of imaging.

Specifically include the following steps:

(1) Select the light source and incident mode according to the spectral characteristics to be measured;

(2) According to the size of the sample to be tested, select a microscopic objective lens with an appropriate magnification; adjust the position of the imaging lens assembly to further determine the magnification, and finally adjust the distance between the microscopic objective lens and the sample to achieve focus;

(3) Set the wavelength scanning range and scanning step of LCTF;

(4) After the LCTF starts to scan, the fluorescence imaging uses the CCD to take pictures at each wavelength until the end of the scan, and the CCD takes microscopic images of the enlarged part of the sample at all wavelengths.

Further, the method also includes the following steps:

After the microscopic image is obtained by the CCD for fluorescence imaging, image processing is performed on the image, including noise reduction processing, and interest points are selected, spectral images are drawn and normalized spectral images are drawn, spectral images are saved and spectral information is output, and the sensory information is obtained. Spectral properties at points of interest.

Specifically, in the step (1), if it is necessary to measure the absorption spectrum, use a wide-spectrum white light source and adopt the transmission incident method; if you need to measure the fluorescence spectrum, use an ultraviolet light source and use the coaxial light input method; Mann spectrum, using 532nm laser light source, adopts coaxial light input method; needs to measure two-photon fluorescence spectrum, then uses infrared laser light source, adopts coaxial light input method; needs to measure infrared spectrum, then uses red light source, adopts coaxial light Either input method or transmissive incidence method is acceptable.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

1. The present invention can simultaneously obtain microscopic imaging and spectral curves at any position in the imaging image by controlling the scanning range and scanning step length of the liquid crystal filter device, can obtain high-quality microscopic imaging quickly and accurately, and can obtain wide-band , high-resolution (1 nm resolution) spectroscopic microscopic images.

2. The position of the imaging lens assembly of the present invention is adjustable in the lens barrel. By changing its up and down position, the imaging size of the sample on the CCD surface is changed, that is, its magnification is changed, so that the whole system can realize continuous magnification of the sample.

3. The present invention only needs to scan the spectrum without scanning the sample (two-dimensional imaging) when performing spectrum shooting, and does not need to perform push-broom and image splicing, and presents two-dimensional images in real time.

4. The present invention can arbitrarily select the part of interest in the image to obtain its spectral characteristic curve, and has wide applicability. Fluorescence spectrum measurement, two-photon fluorescence spectrum measurement, Raman spectrum measurement, and infrared spectrum measurement can be realized for fine tissues by replacing the light source and absorption spectroscopy.

Description of drawings

Fig. 1 is a structural schematic diagram of the device of the present invention.

Fig. 2 is a schematic diagram of the structure of the microscope objective lens used in the embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

As shown in Figure 1, the present embodiment is a kind of microspectral imaging device based on liquid crystal filter device, comprises CCD1 for fluorescence imaging, lens barrel and CCD link head 2, fixing screw 3, lens barrel 4, imaging lens assembly 5, liquid crystal Filter device 6, UV filter 7, coaxial light input hole 8, 45-degree total reflection mirror 9, microscope objective lens 10, X-axis fine-tuning knob 11, sample stage 12, Z-axis fine-tuning knob 13, Y-axis fine-tuning knob 14 And the bracket 15, the device described in this embodiment includes a coaxial incident port, so the light source can be a coaxial light source, and the light source can also be arranged under the sample, so that the light source is a transmission light source. Described fluorescence imaging uses CCD1, lens barrel and CCD link head 2, lens barrel 4, imaging lens assembly 5, liquid crystal filter device 6, ultraviolet filter lens 7, microscope objective lens 10 to be positioned on the same optical axis, 45 degree total reflection The mirror 9 maintains an included angle of 45 degrees with the optical axis, and the coaxial light input hole 8 maintains an included angle of 90 degrees with the optical axis. The position of the imaging lens assembly 5 in the lens barrel 4 is adjustable.

The sample stage 12 is arranged on the base of the support 15, and the base of the support 15 is provided with an X-axis fine-tuning knob 11 and a Y-axis fine-tuning knob 14 for adjusting the horizontal position of the sample stage on the support base. The sample to be tested is placed on the sample stage 12 directly below the microscope objective lens 10, and the liquid crystal filter device 6 is connected with a computer. There is a position-adjustable hook 16 on the bracket 15, and the lens barrel 4 is fixed on this hook 16 by a set screw 3. At the same time, the hook 16 is connected to the bracket 15 and is also provided with a height for adjusting the vertical direction of the hook. Z-axis fine-tuning knob 13. The design of the whole system is flexible, and can be adjusted on the X, Y, and Z axes, and focuses on setting up a coaxial light input system composed of a coaxial light input hole 8 and a 45-degree full reflection mirror 9 and passing through the lens barrel 4 The large-scale adjustable Z-axis adjustment function of moving up and down enables the system light source to adopt various incident modes (transmissive or coaxial, etc.).

The light source may be a gas light source such as a krypton lamp or a xenon lamp, a laser light source, an LED light source, an ultraviolet light source, a tunable light source, or the like. Different light sources can be selected according to different needs, and an appropriate incident mode can be adopted at the same time. The light source can be wide-spectrum white light, a tunable laser, a single-wavelength infrared or ultraviolet laser, or a light wave of a certain monochromatic wavelength, and the wavelength involves the ultraviolet to infrared band. Through different light sources, the system can obtain different spectral imaging. The measurement of absorption spectrum can be realized by using a gas lamp with a relatively wide spectral band. The measurement of fluorescence spectrum can be realized by using ultraviolet lamp or ultraviolet laser. The measurement of two-photon fluorescence spectrum can be realized by using infrared ultrashort pulse laser. For the measurement of Mann spectrum, the measurement of three-dimensional spectrum can be realized by using wavelength-tuned laser.

LCTF with a filter band in the infrared is used for infrared spectrum detection, and an LCTF with a filter band in visible light is used for fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum, and absorption spectrum detection.

The ultraviolet filter 7 is to prevent the ultraviolet light used to excite fluorescence from entering the liquid crystal filter device, specifically a UV mirror with high anti-ultraviolet light and anti-transmission of visible light and infrared spectrum.

The CCD1 for fluorescence imaging is a CCD camera with the characteristics of high sensitivity, high resolution, and fast response time. Infrared CCD for spectrum, or visible-infrared CCD for measuring fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum and absorption spectrum. At the same time, the CCD used for fluorescence imaging adopts the cooling method of liquid nitrogen refrigeration.

The microscopic objective lens 10 is a microscopic objective lens of an achromatic plan infinity system.

A microscopic spectral imaging method based on the above microscopic spectral imaging device, the sample is enlarged through a microscopic objective lens, and the enlarged part of the sample at each wavelength in a certain band is obtained by controlling the scanning range and scanning step of the liquid crystal filter device of imaging.

Specifically include the following steps:

(1) Select the light source and incident mode according to the spectral characteristics to be measured;

(2) According to the size of the sample to be tested, select a microscopic objective lens with an appropriate magnification; adjust the position of the imaging lens assembly to further determine the magnification, and finally adjust the distance between the microscopic objective lens and the sample to achieve focus;

(3) Set the wavelength scanning range and scanning step of LCTF;

(4) After the LCTF starts to scan, the CCD is used for fluorescence imaging to take pictures at each wavelength until the end of the scan, and the CCD captures the microscopic images of the enlarged part of the sample at all wavelengths;

(5) After the microscopic image is obtained by the CCD for fluorescence imaging, image processing is performed on the image, including noise reduction processing, and selecting points of interest, drawing spectral images and normalizing spectral images, and saving spectral images and outputting spectral information , to obtain the spectral properties at the point of interest.

In the step (1), if it is necessary to measure the absorption spectrum, use a broad-spectrum white light source and adopt the transmission incident method; if you need to measure the fluorescence spectrum, use an ultraviolet light source and use the coaxial light input method; if you need to measure the Raman spectrum, Adopt 532nm laser light source, adopt coaxial light input method; need to measure two-photon fluorescence spectrum, use infrared laser light source, adopt coaxial light input method; need to measure infrared spectrum, use red light source, adopt coaxial light input method or Transmissive incident mode is acceptable.

In this embodiment, the exposure time, gain, white balance, etc. of the CCD can be controlled by a computer. Every time the LCTF changes the transmittance, the CCD is excited to take pictures. After the CCD takes pictures, the LCTF is excited again to change the transmittance, and so on until the wavelength scanning is completed.

The image processing can perform noise reduction processing (including image smoothing, image edge detection and image area growth) on the image captured by the CCD, and can select interest points on the picture with the mouse and draw spectral images and normalized spectral images , and save the spectral image and output spectral information.

The operating mechanism of the system is as follows: by selecting a suitable light source and a suitable incident mode, the light is directly applied to the sample to excite the corresponding light (or absorbed light) of the sample, and a certain part of the sample is enlarged through the microscope objective lens, and its The light is shaped into a parallel beam, and the beam enters the liquid crystal filter device (LCTF) through the ultraviolet filter, and the liquid crystal filter device (LCTF) can control the liquid crystal filter device (LCTF). The light wave is filtered out, and the light wave is incident on the CCD array surface through the imaging lens assembly, so as to obtain the imaging (gray image) of the amplified part of the sample at a certain wavelength. By controlling the scanning range and scanning step of the liquid crystal filter device (LCTF), the imaging of the enlarged part of the sample at each wavelength within a certain band can be obtained. Since the intensity of each wavelength of the light generated by the sample is different, the gray scale of the resulting image is also different. Different gray scale values represent the intensity of different wavelengths. Through image processing, a certain point in all images The gray values of the samples are extracted in turn, and the spectral curve of a certain point in the sample image within a certain band can be drawn. The function of obtaining the spectral characteristics of microscopic imaging and samples at the same time is realized, and the spectral characteristics of any point in the image can be selected to be obtained, making spectral analysis more practical and accurate.

The CCD used in this embodiment adopts the MC20 series CCD camera provided by Guangzhou Mingmei, and its main functions are as follows:

a) Sensor (Sensor Type): High-sensitivity 2/3"Sony ICX285Exview HAD CCD, color/black and white optional;

b) Resolution: 1360x1024, 1.4 million pixels;

c) Pixel Size: 6.45μm x6.45μm;

d) Binning Modes 2x2 or 4x4, color;

e) Exposure Control (Exposure Control) 0.5 milliseconds to 40 minutes, automatic exposure;

f) Cooling Type (Cooling Type) is optional, deep four-stage refrigeration to -35 degrees;

g) Real-time Viewing (Real-time Viewing) full-frame real-time preview speed 25 frames per second;

h) Frame Rate (Frame Rate) Full resolution frame rate is 15 frames per second;

i) Digital Interface (Digital Interface) Usb2.0 interface;

j) Shutter Control (Electronic shutter);

The main technical parameters of the liquid crystal filter device (LCTF) used are as follows:

a) Spectral response 400nm-1100nm;

b) Spectral resolution ~ 0.5nm;

c) Spatial resolution: 50 line pairs/mm;

d) Signal-to-noise ratio >40dB;

e) Exposure time 60μs-2000ms;

The vertical effective stroke of the lens barrel is 10mm<H<240mm, the Z-axis fine-tuning accuracy is 0.002mm, the focusing lens can make the imaging zoom continuously from 0.7X to 7X, and the size of the base is 240X180X25(mm) with a movable worktable , the moving distance of the table is 42X42 (mm), the fine-tuning accuracy of X and Y axes is 0.002mm, the microscope objective lens used is Olympus’s 40 times magnification achromatic plan infinity system lens, and the outgoing light is a parallel beam, which is convenient for later The superposition of optical components has both achromatism and flat-field anti-distortion functions. As shown in Figure 2, in this microscope objective, the total length is 48.79mm, the parfocal distance is 45.06mm, and the working distance is 0.6mm. The RMS thread of φ0.800×36 is fixed on the lens barrel. In this embodiment, a high-power ultraviolet laser (argon ion laser) is used as a light source, and a transmission incident mode is adopted. Red blood cell specimens made of quartz glass slides and coverslips are used as samples (quartz glass can transmit ultraviolet light).

Adjust the UV laser to output an average power of 20mW, focus it through a double-convex thin lens with a focal length of 50mm, and then enter the sample to excite its fluorescence. Adjust the appropriate focal length and magnification to obtain a clear microscopic image, and then set up the scan The wavelength is from 400nm to 750nm, the scanning step is 5nm, and the scanning is started, and the microscopic imaging of red blood cells at different wavelengths can be obtained after scanning. When performing image processing, any point on the cell can be selected as the measurement point, and the gray value of the point will be taken from the picture taken at each wavelength, and the spectrum will be drawn with the ordinate as the relative intensity and the abscissa as the wavelength. curve

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. microspectrum imaging device based on liquid crystal filter spare, it is characterized in that, comprise light source, lens barrel, sample stage, fluorescence imaging CCD, support and computing machine, light source is arranged on the relevant position according to incident mode difference, lens barrel is fixed on the support, sample stage is arranged on the bracket base, fluorescence imaging is arranged on lens barrel one end with CCD, and link to each other with computing machine, device also comprises microcobjective, be used for amplify the liquid crystal filter spare that shows that the rear image that forms carries out filtering by microcobjective, the imaging len assembly, fluorescence imaging CCD, lens barrel, the imaging len assembly, liquid crystal filter spare, microcobjective is positioned on the same optical axis, detected sample is placed on the sample stage under the microcobjective, and liquid crystal filter spare links to each other with computing machine.

2. the microspectrum imaging device based on liquid crystal filter spare according to claim 1 is characterized in that, described light source is transmissible optical source, and namely light source is arranged on below the sample.

3. the microspectrum imaging device based on liquid crystal filter spare according to claim 1, it is characterized in that, described light source is coaxial light source, the coaxial light source incidence system is by the axis light input hole, 45 degree total reflective mirrors, microcobjective three parts form, the axis light input hole is arranged on liquid crystal filter spare, on the sidewall perpendicular to the optical axis axis direction between the microcobjective, 45 degree total reflective mirrors are arranged in the light path, 45 degree total reflective mirrors and optical axis keep 45 degree angles, light source enters from the axis light input hole, reflexed to microcobjective and expose to sample by after focusing on by 45 degree total reflective mirrors, wherein be all-trans infrared light and visible light of 45 degree total reflective mirrors.

4. the microspectrum imaging device based on liquid crystal filter spare according to claim 1 is characterized in that, the position of described imaging len assembly in lens barrel is adjustable;

When carrying out adopting the filtering wave band at infrared LCTF when infrared spectrum detects, when carrying out fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum, adopting the filtering wave band at the LCTF of visible light when absorption spectrum detects;

Described fluorescence imaging CCD is in order to measure the infrared CCD of infrared spectrum, perhaps in order to survey fluorescence spectrum, two-photon fluorescence spectrum, Raman spectrum, absorption spectrum visible-infrared CCD;

Described fluorescence imaging adopts the type of cooling of machinery-free shake with CCD;

Described microcobjective is the microcobjective of achromatism flat field infinite distance system.

5. the microspectrum imaging device based on liquid crystal filter spare according to claim 4, it is characterized in that, described device comprises that also the ultraviolet light that prevents for fluorescence excitation enters the ultraviolet filtering mirror of liquid crystal filter spare, this ultraviolet filtering mirror is the UV mirror of the anti-reflection visible light of high anti-ultraviolet light and infrared spectrum, and ultraviolet filtering mirror and microcobjective are positioned on the same optical axis;

Described fluorescence imaging adopts the type of cooling of liquid nitrogen refrigerating with CCD.

6. the microspectrum imaging device based on liquid crystal filter spare according to claim 1, it is characterized in that, the adjustable hook in one position is arranged on the described support, lens barrel is fixed on this hook by fixed screw, and this hook and support connecting place also are provided with one for the Z axis vernier adjustment knob of adjusting hook height in vertical direction simultaneously;

Be provided with on the bracket base for X-axis vernier adjustment knob and the Y-axis vernier adjustment knob of regulating sample stage horizontal direction position on bracket base.

7. microspectrum formation method based on the microspectrum imaging device based on liquid crystal filter spare claimed in claim 1, it is characterized in that, by microcobjective sample is amplified, obtain in a certain wave band the imaging that sample under each wavelength is exaggerated part by the control sweep limit of liquid crystal filter spare and scanning step.

8. the microspectrum formation method based on liquid crystal filter spare according to claim 7 is characterized in that, may further comprise the steps:

(1) spectral characteristic of measuring is as required selected light source and incident mode;

(2) according to the size of detected sample, select the microcobjective of suitable enlargement factor; Be adjusted to the position of picture lens subassembly, further determine enlargement factor, regulate at last microcobjective and sample distance, realize focusing;

(3) wavelength scanning range and the scanning step of setting LCTF;

(4) after LCTF began scanning, fluorescence imaging was taken pictures once under each wavelength with CCD, until the end of scan, CCD photographs that sample is exaggerated micro-image partly under all wavelengths.

9. the microspectrum formation method based on liquid crystal filter spare according to claim 8 is characterized in that, and is further comprising the steps of:

After fluorescence imaging obtains micro-image with CCD, this image is carried out image process, comprise noise reduction process, and choose point-of-interest, draw spectrum picture and normalization spectrum picture, and preserve spectrum picture and output spectrum information, obtain the spectral characteristic at point-of-interest place.

10. the microspectrum formation method based on liquid crystal filter spare according to claim 8 is characterized in that, in the described step (1), needs absorbance spectrum, then uses the white light source of wide spectrum, adopts transmission-type incident mode; Need to measure fluorescence spectrum, then use ultraviolet source, use the axis light input mode; Need to measure Raman spectrum, adopt the 532nm LASER Light Source, adopt the axis light input mode; Need to measure two-photon fluorescence spectrum, then adopt infrared laser light source, adopt the axis light input mode; Need to measure infrared spectrum, then use red-light source, adopt axis light input mode or transmission-type incident mode all can.

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