CN103323410A - A device and a method based on a liquid-crystal filtering device for microscopic spectral imaging - Google Patents
A device and a method based on a liquid-crystal filtering device for microscopic spectral imaging Download PDFInfo
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Abstract
The invention discloses a device and a method based on a liquid-crystal filtering device for microscopic spectral imaging. The device comprises a light source, a lens barrel, a sample bench, a CCD used for fluorescence imaging, a supporter, a computer, a microscopic objective lens, an LCTF used for filtering images formed after being magnified and displayed by the microscopic objective lens, and an imaging lens assembly. The light source is disposed in corresponding positions according to different incidence modes. The lens barrel is fixed to the support. The sample bench is disposed on the pedestal of the support. The CCD is disposed at one end of the lens barrel and connected to the computer. The CCD, the lens barrel, the imaging lens assembly, the LCTF and the microscopic objective lens are disposed at the same optical axis. A sample is disposed on the sample bench that is exactly under the microscopic objective lens. The LCTF is connected to the computer. According to the method provided by the invention, by magnifying the sample utilizing the microscopic objective lens, and by controlling the scanning scope and the scanning increment of the liquid-crystal filtering device, images of magnified parts of the sample at each wavelength in a wave band are obtained. The device and the method are advantageous in that microscopic images of high quality can be obtained rapidly and accurately, magnification can be continuously adjusted, and the device has extensive applicability.
Description
Technical field
The present invention relates to spectral analysis and micro-imaging technique field, particularly a kind of microspectrum imaging device and method based on liquid crystal filter spare.
Background technology
Material is the interaction of light and material to the essence of absorption, emission or scattering that light produces.Study this mechanism, i.e. the variation of light intensity frequency is also drawn corresponding curve and just can be obtained molecular spectrum.Different according to the wavelength coverage of optical radiation and action mode, molecular spectrum can be divided into ultraviolet-visible spectrum, fluorescence spectrum, Raman spectrum, two-photon fluorescence spectrum, infrared spectrum etc.Different spectrum can provide intramolecule different information for the researcher.Spectrum detection technique has the characteristics such as highly sensitive and specificity is good, has become one of mode of commonly using in the modern detecting, is widely used in the fields such as Pharmaceutical Analysis, food hygiene, environment measuring, industrial or agricultural, geologic prospecting and judicial criminal investigation.
As utilize the spectroscopy analysis traditional Chinese medicine ingredients [Zhao Jing, Pang Qichang, Ma Ji, Liu Chuanming, Wang Lin, Cui Daijun, Meng Qingxia, the light spectrum image-forming analytical technology of Chinese Drug Rhizomes of Coptis golden cypress mixed-powder. Acta Optica, 2010,3 (11): 3259-3263].And for example just use liquid crystal wavelengthtunable optical filtering (LCTF) optical spectrum imaging device to carry out preliminary experiment research [the Exline D L of check finger mark streakline as far back as the Claude Roux etc. of Australia in 2003, 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].Along with the progress of photoelectric technology, to update such as light source, imaging device, checkout equipment etc., spectral analysis technique has been deep into the micro-imaging field, and combines with biology and medical science height.As adopt the method for spectral analysis, apoptotic process to cell detect [National Human Genome Research Institute.NHGRI seeks next generation of sequencing technologies.http: //www.genome.gov/12513210.2009,11,4], and for example adopt the FT-IR microspectrum that large gutstring cancer is made cluster analysis [Peter Lascha, Wolfgang Haenschb, Dieter Naumannc, Max Diema, et al.Imaging of colorectal adenocarcinoma using FT-IR micro spectroscopy and cluster analysis[J], Biochimica et Biophysica Acta.2004,1688:176-186], relevant report [the Aikaterini Zoumi that adopts second harmonic and two-photon fluorescence that the arteries micromechanism is observed is also arranged, 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 micro-imaging has developed the detection system of series of new, has expanded biomedical research field and has deepened the depth of investigation to many microstructures.
At present in order to obtain the spectrum picture in the micro-situation, normal operation filter plate, prism and grating spectrograph (monochromator) carry out filtering, thereby obtain image under a certain or some wavelength at the CCD of imaging.In the multispectral camera such as the production of Quest innovations company, the forms such as the optical prism light splitting of employing and optical filter wheel filtering are just arranged, be widely used in remote sensing and the micro-imaging.Adopt filter plate or prism to carry out filtering or light splitting, the spectrum picture that obtains is relatively scattered, is merely able to obtain the imaging under certain several special spectrum, and can not obtain that it is complete, the spectral characteristic in the relative broad range.The various hyperspectral imagers that Specim company releases then are to adopt grating spectrograph to carry out filtering, its wavelength ripple rate scope is long (according to dissimilar, the wavelength span of its filtering is generally more than 500nm), precision high (spectral resolution is below 10nm), but adopt the hyperspectral imager of grating spectrograph+CCD combination, each imaging is a wire (one dimension), obtain the two dimensional image of whole sample, need to push away sample and sweep and take, finally carry out Image Mosaics and form.This way so that in obtaining the process of spectrum picture single need to scanning wavelength, sweep but also will push away sample, imaging time is long, and Image Mosaics also can produce extra noise, so its weak point is also arranged.In order to obtain rapidly, exactly spectrum picture and the corresponding curve of spectrum of sample of two dimension, developed the pattern of liquid crystal filter spare (LCTF)+CCD.The report of the pattern acquisition light spectrum image-forming of relevant employing liquid crystal filter spare (LCTF)+CCD is few, the spectrum picture that mainly concentrates on macro object obtains aspect (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 useful this mode [Xiaoli Sun that blood pH in the blood vessel and kinetic reaction are detected, Yaru Wang, Shangbin Chen, Weihua Luo, Pengcheng Li, Qingming Luo, Simultaneous monitoring of intracellular pH changes and hemodynamic response during cortical spreading depression by fluorescence-corrected multimodal optical imaging[J], NeuroImage.2011,57:873-884].But still lack a whole set of LCTF+CCD system for microscopic system.Subject matter is: at first, for traditional micro optical system, spectrum microscopic system intermediate demand increases many optics original papers, be in the situation that assurance micro-image quality obtains the light spectrum image-forming difficulty larger simultaneously; Secondly, owing to being micro-imaging, its imaging region is very little, the light that namely can enter CCD is very little, when the luminous energy mean allocation is to the CCD face, energy density on its face is very little, and original with regard to fainter fluorescence spectrum signal, two-photon fluorescence spectral signal, raman spectral signal etc. for measuring, its difficulty is quite large.
Therefore, develop a kind of method and relevant device systems that can under microscopic system, fast, accurately obtain spectrum picture, have great significance of scientific research and application value, provide strong support to the application of further expansion spectral analysis on biomedicine.
Summary of the invention
Fundamental purpose of the present invention is to overcome the shortcoming of prior art with not enough, a kind of microspectrum imaging device based on liquid crystal filter spare is provided, this device can obtain high-quality micro-imaging quickly and accurately, only needing when spectrum is taken to carry out the scanning optical spectrum need not scanning samples, applicability be wide.
The present invention also provides a kind of microspectrum formation method based on above-mentioned microspectrum imaging device, the method need not to push away sweeps and Image Mosaics, present in real time two dimensional image, and can directly in image, get a little and obtain the curve of spectrum of this point to point-of-interest.
Purpose of the present invention realizes by following technical scheme: a kind of microspectrum imaging device based on liquid crystal filter spare, 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 liquid crystal filter spare (the Liquid Crystal tunable filter that shows that the rear image that forms carries out filtering by microcobjective, LCTF), 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.
As preferred version, described light source is transmissible optical source, and namely light source is arranged on below the sample.
As another kind of preferred version, described light source is coaxial light source, the coaxial light source incidence system is comprised of axis light input hole, 45 degree total reflective mirrors, microcobjective three parts, the axis light input hole is arranged between liquid crystal filter spare, the microcobjective on the sidewall perpendicular to the optical axis axis direction, 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.
Preferably, the position of described imaging len assembly in lens barrel is adjustable.Thereby can change sample in the imaging size of fluorescence imaging with the CCD the inside by changing its position in lens barrel, namely change its enlargement factor, make whole system can realize continuous amplification to sample.
Preferably, 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.
Further, 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.Thereby avoid ultraviolet light to damage liquid crystal filter spare.
Preferably, the adjustable hook in a 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.Coarse adjustment can be carried out according to object lens to the distance of sample in the position of hook on support, then finely tunes by the Z axis vernier adjustment knob.
As preferably, 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.Thereby can be in the situation that mobile example platform be not adjusted the position that sample is exaggerated by these two vernier adjustment knobs.
Preferably, 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.
Preferably, described fluorescence imaging adopts the type of cooling of machinery-free shake with CCD.Because among the present invention CCD require be highly sensitive, can detect faint light, if adopt air-cooled and water-cooled etc. to have the type of cooling of mechanical shaking, CCD receiving plane array can be affected by mechanical vibration, causes the imaging of imaging len assembly on its face unintelligible, has a strong impact on image quality.
Further, described fluorescence imaging adopts the type of cooling of liquid nitrogen refrigerating with CCD.
Preferably, described microcobjective is the microcobjective of achromatism flat field infinite distance system.Can be the microcobjective of various enlargement factors.Adopt achromatic microcobjective can obtain more accurately image, adopting the infinite distance system is in order conveniently to add various optical elements behind microcobjective, and cancelled the restriction to optical tube length, so that can change at any time different LCTF, different imaging len assembly and different CCD.
A kind of microspectrum formation method based on above-mentioned microspectrum imaging device, 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.
Specifically 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.
Further, described method 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.
Concrete, in the described step (1), need absorbance spectrum, then use the white light source of wide spectrum, adopt 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.
The present invention compared with prior art has following advantage and beneficial effect:
1, the present invention is by the sweep limit of control liquid crystal filter spare and the curve of spectrum that scanning step can obtain optional position in micro-imaging and the image simultaneously, can be quick, accurate etc. obtain high-quality micro-imaging, can obtain in the broadband spectrum micro-image of high precision (resolution is 1nm).
2, the position of the position of imaging len assembly of the present invention in lens barrel is adjustable, by changing its upper-lower position, changes sample in the imaging size of CCD face, namely changes its enlargement factor, makes whole system can realize continuous amplification to sample.
3, the present invention only needing when spectrum is taken to carry out the scanning optical spectrum need not scanning samples (two-dimensional imaging), need not to push away and sweeps and Image Mosaics, presents in real time two dimensional image.
4, the present invention can choose arbitrarily that part interested obtains its spectral pattern in the image, its applicability is wide, can realize micro organization is carried out fluorescence spectral measuring, two-photon fluorescence spectral measurement, raman spectroscopy measurement, infrared spectrometry and absorption spectrum measurement etc. by changing light source.
Description of drawings
Fig. 1 is the structural representation of apparatus of the present invention.
Fig. 2 is the microcobjective structural representation that the embodiment of the invention adopts.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
As shown in Figure 1, a kind of microspectrum imaging device based on liquid crystal filter spare of the present embodiment, comprise fluorescence imaging CCD1, lens barrel and CCD chain joint 2, fixed screw 3, lens barrel 4, imaging len assembly 5, liquid crystal filter spare 6, ultraviolet filtering mirror 7, axis light input hole 8,45 degree total reflective mirrors 9, microcobjective 10, X-axis vernier adjustment knob 11, sample stage 12, Z axis vernier adjustment knob 13, Y-axis vernier adjustment knob 14 and support 15, the described device of the present embodiment has comprised the port of coaxial incident, therefore light source can be coaxial light source, also light source can be arranged on below the sample simultaneously, thereby light source is transmissible optical source.Described fluorescence imaging is positioned on the same optical axis with CCD1, lens barrel and CCD chain joint 2, lens barrel 4, imaging len assembly 5, liquid crystal filter spare 6, ultraviolet filtering mirror 7, microcobjective 10,45 degree total reflective mirrors 9 keep 45 degree angles with optical axis, and axis light input hole 8 keeps 90 degree angles with optical axis.The position of described imaging len assembly 5 in lens barrel 4 is adjustable.
Described light source can be Gas lamp light source, LASER Light Source, led light source, ultraviolet light source, the tunable optical sources etc. such as krypton lamp, xenon lamp.Can select Different Light according to different needs, adopt simultaneously appropriate incident mode.Light source can be the white light of wide spectrum, can be tunable laser, can be the infrared or Ultra-Violet Laser of single wavelength, also can be the light wave of a certain monochromatic wavelength, and wavelength relates to ultraviolet to infrared band.By different light sources, can make system obtain different light spectrum image-formings.Adopt the wider Gas lamp of band, can realize Measurement of absorption spectra, adopt uviol lamp or Ultra-Violet Laser can realize the measurement of fluorescence spectrum, adopt the infrared excess short-pulse laser can realize the measurement of two-photon fluorescence spectrum, adopt 532nm laser can realize the measurement of Raman spectrum, adopt wavelength tuning laser can realize the measurement of three-dimensional spectrum.
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.
Fluorescence imaging CCD1 has high sensitivity, high resolving power, the CCD camera of the characteristics such as the response time is fast, be connected with lens barrel with the CCD chain joint by lens barrel, and link to each other with computing machine, 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.Fluorescence imaging adopts the type of cooling of liquid nitrogen refrigerating with CCD simultaneously.
Described microcobjective 10 is the microcobjective of achromatism flat field infinite distance system.
A kind of microspectrum formation method based on above-mentioned microspectrum imaging device, 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.
Specifically 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;
(5) 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.
In the described step (1), need absorbance spectrum, then use the white light source of wide spectrum, adopt 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.
In the present embodiment, time shutter that can be by computer control CCD, gain, white balance etc.When making a transmitance of the every change of LCTF, excite CCD to take pictures, CCD take pictures finish after, excite again LCTF to change transmitance, so circulation until length scanning finish.
Described image is processed and can be carried out noise reduction process (comprising image smoothing, Image Edge-Detection and image-region growth) to the captured image of CCD, and can choose point-of-interest and draw spectrum picture and the normalization spectrum picture at picture by mouse, and preserve spectrum picture and output spectrum information.
The operating mechanism of described system is as follows: by selecting suitable light source and suitable incident mode the light direct effect is inspired the corresponding light of sample (light that perhaps absorbs) to sample, by microcobjective the sample part is amplified, its light is shaped as parallel beam, this light beam enters liquid crystal filter spare (LCTF) by the ultraviolet filtering mirror, control liquid crystal filter spare (LCTF) can filter out the light wave of a certain wavelength in the light beam (real image after namely being exaggerated) of arrival liquid crystal filter spare, this light wave incides on the ccd array face by the imaging len assembly, thereby the acquisition sample is exaggerated the imaging (gray level image) under a certain wavelength of part.Sweep limit by control liquid crystal filter spare (LCTF) and scanning step just can obtain in a certain wave band the imaging that sample under each wavelength is exaggerated part.Because its each wavelength power of light that sample produces is different, so its gray shade scale of imaging is also different, different gray-scale values are representing the degree of strength of different wave length, process by image certain any gray-scale value in all images is taken out successively, just can depict in certain wave band in the sample image certain any curve of spectrum.Realized obtaining simultaneously the function of the spectral characteristic of micro-imaging and sample, and can select to obtain the spectral characteristic of arbitrfary point in the image, made spectral analysis more practical and accurate.
In the present embodiment the CCD that the adopts MC20 series CCD camera that adopts the bright U.S.A in Guangzhou to provide, its major function is as follows:
A) sensor (Sensor Type): high sensitivity 2/3 " Sony ICX285Exview HAD CCD, colour/black and white is optional;
B) resolution (Resolution): 1360x1024,, 1,400,000 pixels;
C) pixel (Pixel Size) 6.45 μ m x6.45 μ m;
D) pixel mixed mode (Binning Modes) 2x2 or 4x4, colour;
E) exposure control (Exposure Control) is 0.5 millisecond to 40 minutes, automatic exposure;
F) refrigeration type (Cooling Type) is optional, and degree of depth level Four refrigeration is to-35 degree;
G) 25 width of cloth/seconds of live preview (Real-time Viewing) full width live preview speed;
H) 15 width of cloth/seconds of frame rate (Frame Rate) full resolution frame frequency;
I) digital interface (Digital Interface) Usb2.0 interface;
J) shutter control (Shutter Control) electronic shutter (Electronic shutter);
The liquid crystal filter spare that adopts (LCTF) major technique ginseng is as follows:
A) spectral response 400nm-1100nm;
B) spectral resolution～0.5nm;
C) spatial resolution: 50 lines are right/mm;
D) signal to noise ratio (S/N ratio)〉40dB;
E) time shutter 60 μ s-2000ms;
The vertical effective travel of the lens barrel that adopts be 10mm＜H＜240mm, Z axis trimming precision 0.002mm, the condenser lens that adopts can make imaging from 0.7X to the 7X continuous zoom, its size of the base that adopts 240X180X25 (mm) Tape movement worktable, move playing surface apart from 42X42 (mm), X, Y-axis trimming precision 0.002mm, the microcobjective that adopts is that 40 times of Olympus amplify achromatism flat field infinite distance system lens, emergent light is parallel beam, make things convenient for the stack of back optics original paper, have simultaneously the anti-distortion function of achromatism function and flat field, as shown in Figure 2, in this microcobjective, total length is 48.79mm, parfocal distance is 45.06mm, and operating distance is 0.6mm, and the RMS screw thread by φ 0.800 * 36 is fixed on the lens barrel.The present embodiment adopts high-power ultraviolet laser light (Argon ion laser) as light source, and adopts transmission-type incident mode.The red blood cell sample that adopting quartz glass microslide and cover glass are made is as sample (quartz glass is ultraviolet light thoroughly).
It is 20mW that Ultra-Violet Laser is transferred to the output average power, incide sample after focusing on through the focal length biconvex thin lens that is 50mm, inspire its fluorescence, regulate suitable focal length and enlargement factor, obtain clearly micro-imaging, then scanning wavelength is set is 400nm to 750nm, and scanning step is 5nm, and start scanning, can obtain the micro-imaging of red blood cell under different wave length behind the end of scan.Carry out image when processing, can choose that any point is measurement point on the cell, taking out the gray-scale value of this point in the picture that will under each wavelength, take, take ordinate as relative intensity, take horizontal ordinate as wavelength, depicting the curve of spectrum
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within 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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| CN106226247A (en) * | 2016-07-15 | 2016-12-14 | 暨南大学 | A kind of cell detection method based on EO-1 hyperion micro-imaging technique |
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| CN114066818A (en) * | 2021-10-23 | 2022-02-18 | 广州市艾贝泰生物科技有限公司 | Cell detection analysis method, cell detection analysis device, computer equipment and storage medium |
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