CN101241069A - Color dispersion -type multifunctional Hadamard transform microscopical imaging spectrometer - Google Patents
Color dispersion -type multifunctional Hadamard transform microscopical imaging spectrometer Download PDFInfo
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- CN101241069A CN101241069A CNA2008100470251A CN200810047025A CN101241069A CN 101241069 A CN101241069 A CN 101241069A CN A2008100470251 A CNA2008100470251 A CN A2008100470251A CN 200810047025 A CN200810047025 A CN 200810047025A CN 101241069 A CN101241069 A CN 101241069A
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Abstract
A dispersion multifunctional Hadamard conversion micro-imaging spectrograph is provided, comprising an excitation light source, an optical microscope, a lens group, a Hadamard template, a monochrometer and a photoelectric detector. The dispersion multifunctional Hadamard changing micro-imaging spectrograph has a driving mechanism driving Hadamard template; the photoelectric detector is a linear array CCD; the lens group a lens group which focus collimation of photo-signal from the microscope and compresses photo-signal into facula matching size of CCD. The invention integrates functions of fluorescence microscope and microscope Raman spectrometer, bringing Hadamard conversion technology, accomplishing integration for imaging and spectrograph analytical ability on a same meter to obtain sample observing state, spectrum analysis, multi-spectro imaging and image analytical ability. The invention has great system extension and compatibility.
Description
Technical field
The present invention relates to spectral measurement methods and optical image technology field, be specifically related to a kind of color dispersion-type multifunctional Hadamard transform microscopical imaging spectrometer.
Background technology
Fluorescence, Raman light all belong to photoluminescence, and fluorescence spectrum, Raman spectrum can both reflect the composition of material, and can also detection material content according to luminous intensity.Therefore, in the research for fluorescence, Raman light, a variety of instrumentations that have been born, these instruments have produced huge impetus for biomedical research.And brought into play enormous function in scientific research and medical field at fluorescent microscope and these two kinds of instruments of micro-Raman spectroscopy that the research demand of cell and tissue is born.
Common just puts the fluorescent microscope structure as shown in Figure 1.Its structure is made up of Halogen lamp LED 1, condenser 2, diaphragm 3, objective table 4, object lens 5, light splitting piece 6, eyepiece 7, condenser 8, mercury lamp 9 and supporting construction.Sample places on the objective table 4, and the illumination light of Halogen lamp LED 1 sees through condenser 2 and diaphragm 3 sample from the downside irradiation objective table 4, produces the transmission picture.Fluorescence excite general use mercury lamp 9 as excitation source, by color separation filter disc 6 exciting light is reflected object lens 5 and shines the sample surfaces that is positioned on the objective table 4 and produce fluorescence.Light signal sees through color separation filter disc 6 after being entered microscope by object lens 5 collections, enters eyepiece 7.The structures shape of fluorescent microscope fluorescent microscope can only be used to obtain the fluoroscopic image of sample, do not possess spectral analysis capabilities, image does not possess the descriptive power to fluorescence intensity yet.
The micro-Raman spectroscopy structure as shown in Figure 2.Its structure is made up of Halogen lamp LED 1, condenser 2, diaphragm 3, objective table 4, object lens 5, LASER Light Source 6, divided beams device 7, reflective mirror 8, eyepiece 9, trap optical filter 10, Raman spectrometer 11 and supporting construction.Sample places on the objective table 4, and the illumination light of Halogen lamp LED 1 sees through condenser 2 and diaphragm 3 sample from the downside irradiation objective table 4, produces the transmission picture, sees through eyepiece 9 and can observe this picture.The excitation source of micro-Raman spectroscopy generally uses LASER Light Source 6, by beam splitter 7 a branch of superfine laser is seen through object lens and shine the sample surfaces that is positioned on the objective table 4, the Raman signal that produces is collected by object lens 5, draw light signal through divided beams device 7, reflective mirror 8, by trap optical filter 10 filtering Rayleigh scattering lights, enter Raman spectrometer 11 at last then.Micro-Raman system possesses the enlarged image of Raman spectrum analysis ability and sample, but can not obtain the spectrum picture of Raman light.
More than the function of two type systematics separate, but structurally all based on the microscope design, part-structure is similar.There is shared possibility in this part instrument.
The Hadamard transform technology is a kind of modulation technique of similar Fourier transform, has the ability of multi-channel detection and imaging.Adopt this technology can significantly improve signal to noise ratio (S/N ratio).Though this technology has begun to be applied to spectral analysis and micro-imaging field, the instrumental function of using this technology at present is comparatively single, spectral analysis and imaging capability integrated very poor.Disclose the low resolution imaging system of a kind of Hadamard transform as patent ZL94107751.9 (international monopoly Main classification number: G01N21/31 authorizes 1998.7), used 15 * 17 two-dimentional Adama template, obtained the micro-image of 15 * 17 pixels.Patent 01106536.2 discloses a kind of high resolution Adama conversion micro-image analyzer, uses 511 one dimension templates and 512 pixel line array CCDs, has obtained the full resolution pricture of 511 * 512 pixels, but has not possessed the ability of obtaining high-resolution spectra.The report of the spectrometer of relevant application Hadamard transform mainly is to replace the slit of monochromator to be embodied as picture with the Adama template, but possesses the multiple function apparatus appearance as yet of high spectrally resolved ability and better image resolution simultaneously.
Summary of the invention
Purpose of the present invention overcomes above-mentioned the deficiencies in the prior art exactly, a kind of color dispersion-type multifunctional Hadamard transform microscopical imaging spectrometer is provided, this spectrometer can combine with fluorescence spectrum, spectral analysis Raman spectrum and take into account microscope bright-field spectrum and imaging with graphical analysis, thereby realizes multi-functional integrated.
Technical scheme provided by the invention is: the color dispersion-type multifunctional Hadamard transform microscopical imaging spectrometer comprises excitation source, optical microscope, lens combination, Adama template, monochromator and photoelectric detector; Be provided with the driving mechanism that drives the Adama template; Photoelectric detector is a line array CCD; Lens combination will be for focusing on and be compressed into the lens combination of the hot spot that is complementary with the line array CCD size from microscopical light signal collimation.
Above-mentioned Adama template is to be subjected to the direct motor drive screw drive mechanism to drive transmission-type one dimension loop coding template moving linearly, that have one group of light and dark striped.
Above-mentioned monochromator is a reflection grating formula sine mechanism standard monochromator.Rotate the drive grating by motor drive gear, screw rod and rotate conciliation detection wavelength.
Above-mentioned excitation source is high-pressure sodium lamp or laser instrument.
Above-mentioned optical microscope is made of Halogen lamp LED lighting source, the three-dimensional objective table of XYZ, objective lens, eyepiece group and filter filter disc.
Above-mentioned objective lens is made of objective lens support, objective lens and color filter.
Above-mentioned eyepiece group comprises eyepiece support frame and is located at eyepiece on the eyepiece support frame.
Above-mentioned eyepiece support frame is provided with the C interface that is used to connect the hickey of camera and/or CCD is installed and constitutes.
The present invention carries out the function of fluorescent microscope and micro-Raman spectroscopy integrated, introduces the Hadamard transform technology, has successfully realized integrated to imaging and spectral analysis capabilities on same instrument.Obtain sample state observation, spectral analysis, multispectral imaging and image analysis capabilities.
The present invention has good system extension with compatible.This system comparatively comprehensively used to develop take into account fluorescence and Raman, the multi-functional spectrum micro imaging system of spectrum and imaging.And system architecture is suitably simplified, also can obtain laying particular emphasis on the optimization system of a certain function, perhaps simplifying becomes microscopical attached widening parts.
Description of drawings
The optical structure chart of Fig. 1 fluorescent microscope;
The micro-part optical structure chart of the micro-Raman instrument of Fig. 2;
Fig. 3 Hadamard transform fluorescence, Raman spectrum micro imaging system structural representation;
Fig. 4 muti-spectrum imaging principle schematic;
The muti-spectrum imaging of the cancerous issue of Fig. 5 fluorescence probe mark;
The spectral intensity of Fig. 6 fluorescence probe-lighting hours relation curve;
The micro Raman spectra of Fig. 7 Beta Carotene Powder and Raman imaging;
Fig. 8 miniaturization Hadamard transform fluorescent microscopic imaging analyser structural representation;
The Adama fluorescence imaging of Fig. 9 pollen cell and the mensuration of photobleaching.
Embodiment
The Hadamard transform spectrum micro imaging system that the present invention relates to is made up of the software and hardware of microscope, excitation source, Adama template, beam split light path (monochromator), line array CCD detecting device and relevant control.Fig. 3 is the apparatus structure synoptic diagram.
As Fig. 3, object lens 8 collect to comprise transmitted light, fluorescence, Raman light from the various light signals that place the sample on the objective table 6.After light signal entered object lens, the mirror 12 that can be reflected was selected to enter eyepiece 13 so that observe; The perhaps selected spectrum imaging system that enters, light is collimated, focuses on the Adama template 17 by lens 14,15.Light through 17 modulation of Adama template is collimated by lens combination 18, and is focused on the rectangular light spot that becomes suitable entrance slit size at monochromator 21 entrance slit places by cylindrical mirror group 19,20.Enter standard monochromator structure 21 back grating beam splittings, outgoing beam focuses on the quick unit of picture of line array CCD 22.Computer workstation 24 stepper motor controls 23,25, and the signal of reception CCD 22 are by the final related data that obtains of program conversion.Modulated by the Adama template by the light signal that object lens are collected, and use the cylindrical mirror group that light beam is focused on, and use monochromator to carry out beam split to focused light, and use CCD to carry out multi-channel data acquisition, and, export data such as operable spectrum spectrogram, imaging picture, signal intensity by data are decoded.
According to this structure, can select various excitation sources for use, can be mercury lamp or laser.According to application demand, select the light source that different excitation wavelengths and combination of lenses become to be needed.And eliminate the interference of exciting light by optical filter.
According to this structure, can adopt the various lighting systems that excite, as fall to penetrating, transmission, side penetrate etc.Characteristics are per sample used different mode of excitation.For opaque sample, use to fall to penetrating to excite or side is penetrated and excited; For ducted sample, can use laser side-shooting; For transparent sample, can use any mode of excitation comparatively easily.
According to this structure, modulate one by one by the Adama template by the light that microscope is drawn, all generated image by CCD record rear.
According to this structure, through the light of template, by cylindrical mirror focusing becoming a wire hot spot (size is not more than 500 microns * 6000 microns).
According to this structure, the line array CCD size should be complementary with the cylindrical mirror focal beam spot, and line array CCD length and width size should can be obtained by line array CCD with the light of assurance from the outgoing of monochromator exit slit completely more than or equal to focused spot size.
According to this structure, the employed Adama template of Hadamard transform is an one dimension circulation template.Use transparent, opaque corresponding 1 and 0 to generate one group of bar code according to coding rule.The glass sheet that can use etch process processing to be coated with metal is made template, and during Hadamard transform, template should produce required code element to doing the one dimension stepping along coding staff.The step-length of stepping should be identical with coding unit length, and step number should equate with code element number.Also can use liquid crystal panel as template, not need movable platen during conversion, only need to generate required coding bar code successively according to the code element structure.
According to this structure, the length direction pixel count of line array CCD is the Y direction pixel count that generates image, and Adama code symbols number is counted the directions X pixel count that generates image.According to the specific requirement of sample, the present invention can select different image resolution ratios, i.e. 128 * 128 pixels, 256 * 256 pixels and 512 * 512 pixels.
According to this structure, the Adama image of generation is weighed the spectral intensity of each pixel with 256 gray levels.
According to this structure, the Hadamard transform image is the light signal distributed image that derives from sample at certain wavelength place.And the image that area array CCD of installing at the eyepiece position or camera obtain is full spectral signal imaging.
According to this structure, related when the grating rotation of CCD collection and monochromator, promptly obtain the curve of spectrum.
According to this structure,, promptly obtain the time changing curve of light intensity when signal and the time correlation that CCD gathers.
Embodiment 1: Hadamard transform fluorescence, Raman spectrum micro imaging system
Apparatus structure figure as shown in Figure 3, Halogen lamp LED 1, condenser 2, diaphragm 3, objective table 6, object lens 8, light splitting piece 11, reflective mirror 12, eyepiece 13 are the microscopic structure part, laser instrument 4, reflective mirror 5, optical fiber 7, mercury lamp 9, condenser 10 are peripheral excitation source, lens 14,15, diaphragm 16, Adama template 17, lens 18, cylindrical mirror 19,20, monochromator 21 etc. has been set up Adama modulation, beam split light path, line array CCD 22 is a photoelectric detector, and grating control gear 23, computer workstation 24, template control gear 25 etc. are control gear.
To native system, object lens 8 collected light signals can derive from the transmitted light of Halogen lamp LED 1, also can be to be subjected to laser instrument 4, or the resulting luminous signal of mercury lamp 9 excited sample, also come from the non-stimulated luminescence of sample itself.If sample is excited to send fluorescence, then obtain fluorescence spectrum and fluoroscopic image; If sample is excited to send Raman light, then obtain Raman spectrum and Raman signal.Same, transmitted light and luminously can obtain spectrum and imaging equally.
Micro objective 8 is collected required light signal, using catoptron 12 goes out photoconduction, scioptics 14,15 accurately focus on the one dimension circulation Adama template 17, the stepping translation that utilizes stepper motor 25 to control screw rod rotating drive templates realizes the coding to light signal, light behind the coding, through cylindrical mirror 19,20 backs focus on and become an elongated rectangular hot spot, this hot spot is the clear coded image of sample, and be positioned the entrance slit edge of a knife centre of monochromator 21, dispersed light through monochromator 21 beam split, focus on line array CCD 22 surfaces once more, the decoding processing of the signal by 24 couples of CCD of computer workstation 22 output obtains the Adama image in the object lens visual field.
The light that catoptron 12 is derived does not carry out encoding process, scioptics 18 again, cylindrical mirror 19,20 enters monochromator with rectangular light spot later equally, focus on line array CCD (512 pixel) 22 surfaces equally, the grating that workstation 24 control step motors 23 drive 21 li of monochromators rotates, write down the signal from CCD 22 simultaneously, workstation 24 is the stepper motor pulse and the ccd signal data of the correspondence that write down, carries out the spectrum that conversion process obtains the light signal that object lens 8 collect.
Embodiment 2: fluorescence spectrum micro-imaging and spectral analysis
Imaging function and spectral scan function in 1 can realize a kind of muti-spectrum imaging operation in conjunction with the embodiments, and implementation process is as follows:
Use the tissue sample of two kinds of different fluorescence probe marks: use the fluorescence probe labeled cell film of 550nm, use the fluorescence probe labeled cell nuclear of 610nm.Respectively at different wavelength to this tissue sample imaging, consider the vision needs, carry out corresponding pseudo-colours according to wavelength and handle, be among Fig. 4 (1) by a series of images of 530nm to 630nm.Fluorescence spectrum with reference to figure 4 (2).At the spectrum maximal value, select 550 and 610 two width of cloth images, and carry out the data stack by software, final result is Fig. 5.
Fig. 4 (2) is the fluorescence spectrum of sample, has obtained nuclear image (imaging of 610nm place) among Fig. 5 (1), has obtained the image (imaging of 550nm place) of cell membrane among Fig. 5 (2), and Fig. 5 (3) is the superimposed image of Fig. 5 (1) and Fig. 5 (2).Compare with the viewed histofluorescence of eyepiece, the interference that comes from the autofluorescence of cell among Fig. 5 (3) has well been eliminated.
The fluorescence intensity at continuous recording crest place, Fig. 6 is the fluorescence intensity level-time curve of this tissue, has confirmed fluorescence probe under rayed because the phenomenon that photobleaching fails in time gradually.
Embodiment 3: the Raman spectrum of beta carotene and micro-imaging
Use the Raman signal of the laser excitation Beta Carotene Powder of 514nm.Obtain the Raman spectrum of Fig. 7 (1) behind the scanning optical spectrum, at 1000 wave number places, the imaging picture that obtains is Fig. 7 (3).Same position and wavelength place use the transmitted light imaging picture of Halogen lamp LED illumination to be Fig. 7 (2).
Embodiment 4: miniaturization Hadamard transform fluorescent microscopic imaging analyser
This embodiment is primarily aimed at the more single fluorescence imaging of spectral component, and its optical texture synoptic diagram as shown in Figure 8.
By reducing the requirement to spectrally resolved ability, the miniaturization of the instrument of realization.Use catoptron 12,20 in light path, when shortening light path light path is folded, and use less lens 11,13,16,17,19 have constituted the beam split light path of comparatively simplifying with small-sized grating 18 instruments, long-pending with the compressometer body.The Adama template 15 same one dimension 511 code element circulation templates of using, the CCD 21 same 512 pixel line array CCDs that use.Control gear has been done corresponding simplification, but structure still meets Fig. 3.
The exciting light that mercury lamp 8 sends, by object lens 5, shone the sample surfaces on the objective table 4 by color separation filter 6 reflection backs, the fluorescence that sends is collected by object lens 5, mirror 9 turns to and focused on Adama template 15 surfaces by lens 11,13 through being reflected behind the color separation filter 6.By the light behind one dimension Adama template 15 codings, by grating 18 beam split, the dispersed light after the beam split is focused on line array CCD 21 surfaces by lens 19 by lens combination 16-17 collimation back.The data acquisition with CCD is moved in the stepping of workstation control Adama template 15, and can obtain fluoroscopic image by decoding.
Grating distinguishing ability in the present embodiment is relatively poor, mainly plays simple color filtration, and some spectra part is strengthened in imaging.As required, also can replace the beam split light path with optical filter.
Embodiment 5: the Hadamard transform fluoroscopic image is analyzed
The beautiful curtain pollen that collects is dyeed with acridine orange (AO), use the blue-light excited of mercury lamp, the fluoroscopic image of the beautiful curtain pollen cell that obtains is shown in Fig. 9 (1), a among the figure, b, the fluorescence intensity of three cells of c and the big I of cell are accurately measured by analysis software, and its fluorescence intensity level is respectively 559.5k, 633.9k, 432.4k, maximum gauge is respectively 49.8,48.0,50.4 μ m, and minimum diameter is respectively 33.0,35.4,32.4 μ m.Per minute carries out Polaroidly to three pollen cells in 20 minutes, and the quantitative data in each width of cloth image is analyzed, and obtains Fig. 9 (2).Fig. 9 (2) has shown fluorescence intensity-time curve of pollen cell b, has reflected pollen fluorescence under rayed exactly because the phenomenon that photobleaching fails in time gradually.
Claims (9)
1. the color dispersion-type multifunctional Hadamard transform microscopical imaging spectrometer comprises excitation source, optical microscope, lens combination, Adama template, monochromator and photoelectric detector, it is characterized in that: be provided with the driving mechanism that drives the Adama template; Photoelectric detector is a line array CCD; Lens combination will be for focusing on and be compressed into the lens combination of the hot spot that is complementary with the line array CCD size from microscopical light signal collimation.
2. spectrometer according to claim 1 is characterized in that: the Adama template is to be subjected to the transmission-type one dimension loop coding template drive mechanism moving linearly, that have one group of light and dark striped.
3. spectrometer according to claim 1 and 2 is characterized in that: monochromator is a reflection grating formula sine mechanism standard monochromator.
4. spectrometer according to claim 3 is characterized in that: monochromator is provided with and drives the driving mechanism that grating rotates.
5. spectrometer according to claim 1 and 2 is characterized in that: excitation source is high-pressure sodium lamp or laser instrument.
6. spectrometer according to claim 1 and 2 is characterized in that: optical microscope is made of Halogen lamp LED lighting source, the three-dimensional objective table of XYZ, objective lens, eyepiece group and filter filter disc.
7. spectrometer according to claim 6 is characterized in that: objective lens is made of objective lens support, objective lens and color filter.
8. spectrometer according to claim 6 is characterized in that: the eyepiece group comprises eyepiece support frame and is located at eyepiece on the eyepiece support frame.
9. spectrometer according to claim 8 is characterized in that: eyepiece support frame is provided with the C interface that is used to connect the hickey of camera and/or CCD is installed and constitutes.
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