CN103278919A - Confocal microscopic imaging method for colored three-dimensional area - Google Patents
Confocal microscopic imaging method for colored three-dimensional area Download PDFInfo
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Abstract
The invention relates to a confocal microscopic imaging method for a color three-dimensional area. The method comprises the following steps of building an NiN channel wavelength division frequency division modulation laser light path and microscopic imaging of a wavelength division frequency division multiplexing confocal microscope, splitting a white light super-continuum spectrum via a holographic polymer dispersed liquid crystal grating array, and focusing light spectrums of different wavebands at different positions on an axis via a microscope array to realize Z-axis detection; meanwhile, focusing light spectrums of the same waveband at different positions on an X-Y plane via the grating array, driving the polymer dispersed liquid crystal grating array through a driving circuit, and chopping the light spectrums of the same waveband to increase the transverse resolution of a sample and realize multipoint parallel real-time detection; and enabling a certain point of the sample to be detected by light spectrums of different wave bands by applying a zooming microobjective and changing the focal length of the microobjective, comparing images received after every change of the focal length to judge the color of certain point of the sample, and analyzing acquired information to construct a three-dimensional detection effect image with color information.
Description
Technical field
The present invention relates to a kind of micro-imaging technique, particularly the regional confocal microscopic imaging method of a kind of color solid.
Background technology
Confocal microscope has had tremendous development so far from producing, progress along with science and technology, confocal microscope miscellaneous is developed out, when scan mode more and more tends to modern science and technologyization, the detection information receiving system is also more and more accurate, make instrument can survey littler more accurate sample, also satisfied the requirement in epoch.
The confocal scanning microscope technology is the novel microtechnic that integrates novel confocal principle, laser scanner technique and computer image processing technology, is a kind of typical new and high technology photoelectric instrument.Compare with simple microscope, have higher lateral resolution and axial resolution preferably, and can obtain a large amount of faultage images by the object different depth is successively scanned, by the synthetic three-dimensional image of Computer Processing.Compare with electron microscope technique, can cut into slices to sample, dynamic observe thereby can keep the living body sample, thereby all be widely used in fields such as biology, medical diagnosis, industry, agricultural, national defence, scientific research, life sciences.
At present, the ultrawhite light that has occurred using photonic crystal fiber to produce is made colored confocal microscope, the 3-dimensional digital confocal Raman microscope of exciting light source and the optical fiber novel microscopes such as multiplexed confocal microscope that are coupled, the laboratory has worked out multi-path frequency-division duplicating fluorescent confocal microscopic system, adopt coaxial electric control zooming Polymer Dispersed Liquid Crystal, realize hyperchannel frequency-division multiplexing fluorescent confocal method, but can only survey sample plane information at present, can't realize three-dimensional detection.At present, the confocal microscope of detection of three dimensional information is arranged also, but owing to use mechanical copped wave, system bulk is very big, and can not realize three-dimensional measurement truly, on resolution and speed of detection, has problem to exist.
Summary of the invention
The present invention be directed to the problem that does not have the confocal microscope of detection of three dimensional information truly at present, a kind of color solid zone confocal microscopic imaging method has been proposed, propose to adopt electric control zooming Polymer Dispersed Liquid Crystal grating, when realizing frequency division multiplexing, realize the confocal microscopic imaging method of wavelength-division multiplex, realize the real-time three-dimensional of sample is surveyed.This system adopts holographic polymer dispersed liquid crystal grating array and microlens array, has the integrated form design, is convenient to control, has also reduced mechanical noise and machine volume, has improved resolution and the speed of confocal microscope.
Technical scheme of the present invention is: a kind of color solid zone confocal microscopic imaging method specifically comprises the steps:
1) build N í N channel wave and divide frequency division modulated laser light path: with femto-second laser, photonic crystal fiber, beam expanding lens, H-PDLC grating array, colour killing loose microlens array, beam splitter, condenser lens, the telescope imaging system formed by lens and zoom microcobjective array coaxial building successively, focus on the sample, the femto-second laser pulse that femto-second laser is sent is coupled to and obtains white light super continuum light spectrum in the photonic crystal fiber, is coupled into beam expanding lens again; The light beam of beam expanding lens outgoing arrives the modulation of carrying out light splitting and different frequency on the N í N formula H-PDLC grating array, the light beam that light splitting modulates is by the diffusing microlens array of colour killing, being adjusted into parallel beam penetrates, form the parallel beam that different-waveband spectrum distributes from top to bottom successively in the space, make a parallel beam part see through by beam splitter, part reflection, the parallel beam that sees through beam splitter focuses on the axle through condenser lens, and focused beam focuses on the sample through lens and zoom microcobjective array;
2) build the micro-imaging of Wave-division frequency division multiplex confocal microscope: the telescope imaging system of the light beam that sample reflects through being constituted by lens and zoom microcobjective array, form parallel beam behind the line focus lens again, enter into by the diffusing object lens of colour killing through beam splitter reflection, the filtering system that filter band and object lens are formed, wherein the focus of the diffusing object lens of colour killing and the focus of object lens all are positioned at filter band central small hole place, filtered parallel beam arrives beam splitter, a part sees through and is received by ccd video camera, another part is by beam splitter reflection, and PMT receives by photomultiplier;
3) signals collecting and processing: ccd video camera collected specimens information, and three-dimensional information is shown in the plane, the grating fringe level is placed or is placed perpendicular to platform in platform, distributed with from top to bottom order or order from left to right by the different-waveband spectrum after the light splitting of N í N formula H-PDLC grating array accordingly, just can separately record depth information and the plane information of sample, be about to three-dimensional information and convert plane information to and carry out record, send in the computing machine and carry out three-dimensional structure according to the sample message that has detected; Focus by zoom microcobjective array, zoom each time, obtain a kind of spectrographic detection result of wave band, by repeating the focusing operation, can access the result of detection of the spectrum of different-waveband in this position, by the resulting image information of ccd video camera after the contrast focusing each time, find out the most weak spectrum of light intensity that this position sensing arrives, then this spectrum is the color of this position of sample, detection image to each position of sample all compares, and then can obtain the colouring information of sample; Photomultiplier PMT will detect the light intensity signal summation of a depth direction on the sample and carry out opto-electronic conversion, by A/D converter signal is transported to computing machine, spectrum with different frequency is carried out demodulation, carry out demodulation by frequency separately, modulation signal and the cosine signal with same carrier frequencies are multiplied each other, again by the unnecessary frequency spectrum of low-pass filter elimination, can obtain original signal, namely obtain the depth information of the sample that spectrum that this depth direction of sample namely has same frequency surveys.
N í N formula H-PDLC grating array is made up of N H-PDLC grating of N í in the described step 1), each H-PDLC grating is to be formed by polymkeric substance and liquid crystal alternate combinations, by being interfered, the PDLC material forms holographic grating between two glass plates that have an ITO film, the H-PDLC grating is placed with the grating fringe direction direction parallel with surface level, each H-PDLC grating of white light continuous spectrum process is by light splitting, the spectrum of the different-waveband after the light splitting is separated from top to bottom successively, and the spectrum after the light splitting is through being focused onto the different depth position of sample one direction behind the optical system device; Regulate driving circuit, light signal is modulated into square-wave signal, triangular signal, sinusoidal signal, to the copped wave of different-waveband spectrum, make the spectrum of same wave band have different frequencies, then through being focused onto the diverse location on a plane of sample behind the optical system device, the H-PDLC grating array carries out light splitting and chopping modulation to the white light continuous spectrum.Described to setting different chopping frequencies respectively on N H-PDLC grating of each N í, each frequency values satisfies the Nyquist sampling theorem, must not be higher than half of sample frequency, simultaneously, the carrier frequency of any two signals and their difference must be more than or equal to the twices of highest signal frequency.
Colour killing is loose microlens array by can cancellation optical grating diffraction one-level forming with the diffusing semi-permeable mirror N í N formula array of the colour killing of external spectrum in the described step 1), regulate the focus of each semi-permeable mirror of the diffusing microlens array of colour killing, be located at it each H-PDLC grating place of corresponding H-PDLC grating array, the light beam that light splitting modulates is by the diffusing microlens array of colour killing, be adjusted into parallel beam and penetrate, form the parallel beam that different-waveband spectrum distributes from top to bottom successively in the space.
Described step 1) and 2) in zoom microcobjective array by with N í N formula H-PDLC grating array in each H-PDLC grating one by one corresponding magnification be that 40 times, numerical aperture are that 0.65 zoom microcobjective is formed.
Described step 2) filter band central small hole diameter equals Aili spot when size in, namely R=
, R is little pore radius, and D is the clear aperature of object lens, and F is the focal length of the diffusing object lens of colour killing.
Beneficial effect of the present invention is: color solid of the present invention zone confocal microscopic imaging method, wavelength-division multiplex is combined with frequency division multiplexing, sample is carried out three-dimensional probe, obtain sample message in real time, and the information that obtains analyzed, three three-dimensional probe design sketchs can be made up, the colouring information of sample can be accessed simultaneously, more quick more, efficient than at present general confocal micro detection system.
Description of drawings
Fig. 1 is the light path synoptic diagram of color solid of the present invention zone confocal microscopic imaging method;
Fig. 2 is N í N formula H-PDLC grating array synoptic diagram of the present invention;
Fig. 3 is the diffusing microlens array synoptic diagram of N í N formula of the present invention colour killing;
Fig. 4 is N í N formula zoom microcobjective array synoptic diagram of the present invention;
Fig. 5 gathers the image information synoptic diagram for CCD of the present invention;
Fig. 6 is filter band synoptic diagram of the present invention.
Embodiment
Color solid zone confocal microscopic imaging method is specifically addressed:
A: build N í N channel wave and divide frequency division modulated laser light path: light path synoptic diagram as shown in Figure 1, with femto-second laser 1, photonic crystal fiber 2, beam expanding lens 3, H-PDLC grating array 4, colour killing loose microlens array 5, beam splitter 6, condenser lens 7, the telescope imaging system formed by lens 8 and zoom microcobjective array 9 coaxial building successively.Axis and platform remain level.At first the femto-second laser pulse that femto-second laser 1 is sent is coupled to and obtains white light super continuum light spectrum in the photonic crystal fiber 2, is coupled into beam expanding lens 3 again; The light beam of beam expanding lens 3 outgoing arrives the modulation of carrying out light splitting and different frequency on the N í N formula H-PDLC grating array 4, N í N formula H-PDLC grating array synoptic diagram as shown in Figure 2, formed by N H-PDLC grating 19 of N í, each H-PDLC grating 19 is to be formed by polymkeric substance 20 and liquid crystal 21 alternate combinations, by being interfered, the PDLC material forms holographic grating between two glass plates that have an ITO film, H-PDLC grating 19 is placed with the grating fringe direction direction parallel with surface level, each H-PDLC grating 19 of white light continuous spectrum process is by light splitting, and because H-PDLC grating 19 stripe directions are parallel to surface level, the spectrum of the different-waveband after the light splitting is separated from top to bottom successively, spectrum after the light splitting is through being focused onto the different depth position of sample 10 a certain directions 24 behind the optical system device, thereby can survey the depth information of sample 10 a certain depth directions 24, because H-PDLC grating array 4 is made up of N H-PDLC grating 19 of N í, then the spectral distribution of the different-waveband after the light splitting is in whole space.Can regulate driving circuit, light signal is modulated into square-wave signal, triangular signal, sinusoidal signal or other signal, generally speaking, the light signal of continuous strength can be modulated into square-wave signal, thereby to the copped wave of different-waveband spectrum, make the spectrum of same wave band have different frequencies, then through being focused onto the diverse location on sample 10 some planes 25 behind the optical system device, thereby can survey the information on sample 10 some planes 25, H-PDLC grating array 4 pairs of white light continuous spectrums have played the effect of light splitting, chopping modulation.The colour killing microlens array synoptic diagram that looses as shown in Figure 3, except the diffusing semi-permeable mirror 22 of the colour killing that forms array, be lighttight in the diffusing microlens array 5 of colour killing, because we only utilize grating diffration one-level light to experimentize, select for use colour killing loose semi-permeable mirror 22 beyond can cancellation optical grating diffraction one-level spectrum and because the influence of the transmitted light that produces when powering up for H-PDLC grating array 4, regulate the focus of each semi-permeable mirror 22 of the diffusing microlens array 5 of colour killing, be located at it each H-PDLC grating 19 place of corresponding H-PDLC grating array 4, the light beam that light splitting modulates is by the diffusing microlens array 5 of colour killing, to be adjusted into parallel beam through the light beam of the focusing of H-PDLC lens arra 4 outgoing and penetrate, form the parallel beam that different-waveband spectrum distributes from top to bottom successively in the space.Make a parallel beam part see through by beam splitter 6, part reflection, the parallel beam that sees through beam splitter 6 focuses on the axle through condenser lens 7, focused beam is 40 times via lens 8 and magnification, numerical aperture is the telescope imaging system that 0.65 zoom microcobjective array 9 is formed, zoom microcobjective array synoptic diagram as shown in Figure 4, zoom microcobjective array 9 by with N í N formula H-PDLC grating array 4 in each H-PDLC grating 19 one by one corresponding magnification be 40 times, numerical aperture is 0.65 zoom microcobjective 23 compositions, to converge to respectively on the sample 10 through the spectrum of the different-waveband of lens 8, CCD gathers the image information synoptic diagram on the sample as shown in Figure 5, because zoom microcobjective array 9 is made up of N zoom microcobjective 23 of N í, realization converges to the spectrum of different-waveband the different depth of sample 10 a certain depth directions 24, and same band spectrum, because its frequency difference, then be converged onto on the sample 10 a certain planes 25, the final three-dimensional detection of realizing, can regulate simultaneously the focal length of zoom microcobjective 23, a certain position for sample 10, zoom each time, obtain a kind of spectrographic detection result of wave band, by repeating the focusing operation, can access the result of detection of the spectrum of different-waveband in this position, the detection image that obtains by contrast, thereby can obtain the color of sample 10 some positions, then can find out the colouring information of sample.
B: the micro-imaging of building the Wave-division frequency division multiplex confocal microscope: the light beam that is reflected by sample 10 is through being that 40 times, numerical aperture are that telescope imaging system and condenser lens 7 backs that 0.65 zoom microcobjective array 9 constitutes form parallel beams by lens 8 and magnification, enter into the filtering system of being formed by the diffusing object lens 11 of colour killing, filter band 12 and object lens 13 through beam splitter 6 reflections, wherein the focus of the diffusing object lens 11 of colour killing and the focus of object lens 13 all are positioned at filter band 12 aperture centers, and the method is aperture filtering.At the center of filter band 12 aperture 26 is arranged, is used for the elimination veiling glare, filter band synoptic diagram as shown in Figure 6, the aperture size is more near ideal point, then resolution is more high, image quality is more good, when the theoretical diameter that calculates aperture 26 equals the Aili spot size, namely R=
, R is little pore radius, and D is the clear aperature of object lens 13, and F is the focal length of the diffusing object lens 11 of colour killing, can guarantee high resolving power and the chromatography ability of confocal imaging like this, has enough light to be detected device by aperture again and receives.Filtered parallel beam arrives beam splitter 14, a part sees through and is received by ccd video camera 15, signal by ccd video camera 15 collected specimens, in computer software, obtain the sample 3-D view at last, and come the colouring information of judgement sample by contrast ccd video camera 15 received images, another part is reflected by beam splitter 14, and PMT16 receives by photomultiplier.The lateral resolution r of this confocal microscope and axial resolution z are expressed as respectively: r=
, z=
, wherein, λ is the wavelength that sample reflects, NA represents the numerical aperture of zoom microcobjective 23,
Be zoom microcobjective refraction coefficient, temporal resolution is relevant with chopping frequency.
C: based on making and the characteristic of Polymer Dispersed Liquid Crystal grating array light splitting chopping modulation: as the key component of color solid zone confocal microscopic imaging design, N í N formula H-PDLC grating array 4 can be realized the function of light splitting, frequency modulation simultaneously.H-PDLC grating array synoptic diagram as shown in Figure 2, H-PDLC grating array 4 is made up of N H-PDLC grating 19 of N í, each H-PDLC grating 19 is to be combined by polymkeric substance 20 and liquid crystal 21, forms holographic grating by the PDLC material is interfered between two glass plates that have an ITO film.H-PDLC grating 19 is placed with the grating fringe direction direction parallel with surface level, each H-PDLC grating 19 of white light continuous spectrum process is by light splitting, and because H-PDLC grating 19 stripe directions are parallel to surface level, the spectrum of the different-waveband after the light splitting is separated from top to bottom successively, spectrum after the light splitting is through being focused onto the different depth position of sample 10 a certain directions 24 behind the optical system device, thereby can survey the depth information of sample 10 a certain depth directions 24, because H-PDLC grating array 4 is made up of N H-PDLC grating 19 of N í, then the spectral distribution of the different-waveband after the light splitting is in whole space.Can regulate driving circuit, light signal is modulated into square-wave signal, triangular signal, sinusoidal signal or other signal generally speaking, can be modulated into square-wave signal with the light signal of continuous strength, thereby to the copped wave of different-waveband spectrum, make the spectrum of same wave band have different frequencies.Each H-PDLC grating 19 in the N í N H-PDLC grating array 4 is set different chopping frequencies respectively, each frequency values should satisfy the Nyquist sampling theorem, must not be higher than half of sample frequency, simultaneously, the carrier frequency of any two signals and their difference must be more than or equal to the twices of highest signal frequency, prevent from producing between each passage and crosstalk, thereby influence testing result, then through being focused onto the diverse location on sample 10 some planes 24 behind the optical system device, thereby can survey the information on sample 10 some planes 25,4 pairs of white light continuous spectrums of H-PDLC grating array have played light splitting, the effect of chopping modulation.
D: based on the focusing characteristic of zoom microcobjective array: as the key component of color solid zone confocal microscopic imaging design, N í N formula zoom microcobjective array 9 has the focusing function.Zoom microcobjective array synoptic diagram as shown in Figure 4, zoom microcobjective array 9 by with N í N formula H-PDLC grating array 4 in each H-PDLC grating 19 one by one corresponding magnification be that 40 times, numerical aperture are that 0.65 zoom microcobjective 23 is formed, will converge to respectively on the sample 10 through the spectrum of the different-waveband of lens 8.Because zoom microcobjective array 9 is made up of N zoom microcobjective 23 of N í, realization converges to the spectrum of different-waveband the different depth of sample 10 a certain depth directions 24, and same band spectrum, because its frequency difference, then be converged onto on the sample 10 a certain planes 25, finally realize three-dimensional detection; Can regulate simultaneously the focal length of zoom microcobjective 23, a certain position for sample 10, zoom each time, obtain a kind of spectrographic detection result of wave band, by repeating focusing operation, can access the result of detection of the spectrum of different-waveband in this position, the detection image that obtains by contrast, thereby can obtain the color of sample 10 some positions, then can find out the colouring information of sample.
E: the realization of signals collecting and processing section: ccd video camera 15 collected specimens information, and three-dimensional information is shown in the plane, the grating fringe level is placed or is placed perpendicular to platform in platform, distributed with from top to bottom order or order from left to right by the different-waveband spectrum behind the H-PDLC grating beam splitting accordingly, just can separately record depth information and the plane information of sample.If the grating fringe level is placed in platform, different spectrum behind grating beam splitting spatially distribute from top to bottom so, like this, the spectrum of different colours disperses in the vertical direction, the different colours information that ccd video camera 15 receives at vertical plane is the depth information of a certain depth direction 24 of sample 10, owing to be N í N formula H-PDLC grating array 4, ccd video camera 15 will record in the plane successively through the information of each passage of every kind of spectrum of ovennodulation again, the information that the same color of record is positioned at diverse location is the information on sample 10 a certain planes 25, formed and convert three-dimensional information to plane information and carry out record, carried out three-dimensional structure according to the sample message that has detected on computers.Utilize the characteristic of zoom microcobjective 23 simultaneously, for example for a certain position of sample 10, zoom each time, obtain a kind of spectrographic detection result of wave band, by repeating the focusing operation, can access the result of detection of the spectrum of different-waveband in this position, by ccd video camera 15 resulting image informations after the contrast focusing each time, find out the most weak spectrum of light intensity that this position sensing arrives, then this spectrum is the color of sample 10 a certain positions, detection image to each position of sample 10 all compares, and then can obtain the colouring information of sample, draws the sample 3 d effect graph.Photomultiplier PMT16 carries out opto-electronic conversion with the light intensity signal summation of a certain depth direction 24 of the sample that detects, by A/D converter 17 signal is transported to computing machine 18, spectrum with different frequency is carried out demodulation, carry out demodulation by frequency separately, modulation signal and the cosine signal with same carrier frequencies are multiplied each other, again by the unnecessary frequency spectrum of low-pass filter elimination, can obtain original signal, namely obtain the depth information of the sample that spectrum that the some depth directions 24 of sample namely have same frequency surveys, improved the lateral resolution of system.
Color solid zone confocal microscopic imaging is the confocal microscopic imaging method that combines wavelength-division multiplex technique and frequency multiplexing technique.The present invention makes the light splitting of white light super continuous spectrums by the holographic polymer dispersed liquid crystal grating array, because the existence of aberration, the spectrum of different-waveband is different through the position that the microcobjective array focuses on the axle, thereby realizes the detection of Z axle, i.e. depth finding; Simultaneously, pass through grating array, make the spectrum of same wave band focus on the diverse location of X-Y plane, can drive the Polymer Dispersed Liquid Crystal grating array by driving circuit, spectrum to same wave band carries out copped wave, improve the lateral resolution of sample, because what use is N í N formula grating array, can realize the multiple spot real-time detection that walks abreast; Used the zoom microcobjective, made that by the focal length that changes object lens certain of sample a bit can be by the spectrographic detection of different-waveband, contrast is each to change the color that the received image of focal length comes the judgement sample point, can realize like this colour of sample is surveyed; Wavelength-division multiplex is combined with frequency division multiplexing, sample is carried out three-dimensional probe, obtain sample message in real time, and the information that obtains analyzed, can make up three three-dimensional probe design sketchs, can access simultaneously the colouring information of sample, more quick more, efficient than at present general confocal micro detection system.
This patent has designed color solid zone confocal micro imaging system.The main composition of system comprises: the femto-second laser pulse that femto-second laser is sent in employing is coupled to the white light super continuum light spectrum that obtains in the high non-linear photon crystal optical fiber, N í N formula Polymer Dispersed Liquid Crystal grating array, the colour killing microlens array that looses, the semi-transparent beam splitter that partly returns, magnification is 40 times zoom microcobjective array, high-resolution color ccd video camera and photomultiplier PMT.In addition, also comprise condenser lens and the lens that effects such as focusing, collimation, amplification are arranged in light path.
The light path synoptic diagram of the color solid zone confocal microscopic imaging method of building as shown in Figure 1, at first the femto-second laser pulse that femto-second laser 1 is sent is coupled to and obtains white light super continuum light spectrum in the photonic crystal fiber 2, be coupled into beam expanding lens 3, the light beam of beam expanding lens 3 outgoing arrives the modulation of carrying out light splitting and different frequency on the N í N formula H-PDLC grating array 4, the light beam that light splitting modulates to be by the colour killing microlens array 5 that looses, and will be adjusted into parallel beam through the light beam of the focusing of H-PDLC grating array 4 outgoing and penetrate.Make a parallel beam part see through by beam splitter 6, part reflection, the parallel beam that sees through beam splitter 6 focuses on the axle through condenser lens 7, focused beam is 40 times via lens 8 and magnification, numerical aperture is that the telescope imaging system that 0.65 zoom microcobjective array 9 is formed focuses on sample 10, the light beam that is reflected by sample 10 is through being that telescope imaging system and condenser lens 7 backs that 40 times zoom microcobjective array 9 constitutes form parallel beams by lens 8 and magnification, enter into by the diffusing object lens 11 of colour killing through beam splitter 6 reflections, the filtering system that filter band 12 and object lens 13 are formed, filtered parallel beam arrives beam splitter 14, a part sees through and is received by ccd video camera 15, signal by ccd video camera 15 collected specimens, in computer software, obtain the sample 3-D view at last, and come the colouring information of judgement sample by contrast ccd video camera 15 received images, draw the sample 3 d effect graph, another part is reflected by beam splitter 14, PMT16 receives by photomultiplier, by A/D converter 17 signal is transported to computing machine 18, carries out demodulation.
System adopts the femto-second laser pulse that is sent by femto-second laser to be coupled in the high non-linear photon crystal optical fiber, the white light super continuum light spectrum that obtains is as light source, adopt N í N formula H-PDLC grating array to realize light splitting and copped wave, can realize the Z axle of sample and the real-time detection of X-Y plane, realized three-dimensional detection, utilize the characteristic of zoom lens simultaneously, make the spectrum of different-waveband can detect same position, by the contrast image that obtains of surveying, obtain the colouring information of sample at last, and have characteristics such as high spatial resolution and temporal resolution.
Claims (6)
1. a color solid zone confocal microscopic imaging method is characterized in that, specifically comprises the steps:
1) builds N í N channel wave and divide frequency division modulated laser light path: with femto-second laser (1), photonic crystal fiber (2), beam expanding lens (3), H-PDLC grating array (4), the colour killing microlens array (5) that looses, beam splitter (6), condenser lens (7), the telescope imaging system of being made up of lens (8) and zoom microcobjective array (9) is coaxial building successively, focus on the sample (10), the femto-second laser pulse that femto-second laser (1) is sent is coupled to and obtains white light super continuum light spectrum in the photonic crystal fiber (2), is coupled into beam expanding lens (3) again; The light beam of beam expanding lens (3) outgoing arrives the modulation of carrying out light splitting and different frequency on the N í N formula H-PDLC grating array (4), the light beam that light splitting modulates is by the diffusing microlens array of colour killing, being adjusted into parallel beam penetrates, form the parallel beam that different-waveband spectrum distributes from top to bottom successively in the space, make a parallel beam part see through by beam splitter (6), part reflection, the parallel beam that sees through beam splitter (6) focuses on the axle through condenser lens (7), and focused beam focuses on the sample (10) through lens (8) and zoom microcobjective array (9);
2) build the micro-imaging of Wave-division frequency division multiplex confocal microscope: the light beam that sample (10) reflects is through the telescope imaging system by lens (8) and zoom microcobjective array (9) formation, line focus lens (7) back forms parallel beam again, enter into by the diffusing object lens (11) of colour killing through beam splitter (6) reflection, the filtering system that filter band (12) and object lens (13) are formed, wherein the focus of the diffusing object lens (11) of colour killing and the focus of object lens (13) all are positioned at filter band (12) central small hole place, filtered parallel beam arrives beam splitter (14), a part sees through and is received by ccd video camera (15), another part is by beam splitter (14) reflection, by photomultiplier PMT(16) receive;
3) signals collecting and processing: ccd video camera (15) collected specimens information, and three-dimensional information is shown in the plane, the grating fringe level is placed or is placed perpendicular to platform in platform, distributed with from top to bottom order or order from left to right by the different-waveband spectrum after N í N formula H-PDLC grating array (4) light splitting accordingly, just can separately record depth information and the plane information of sample, be about to three-dimensional information and convert plane information to and carry out record, send in the computing machine and carry out three-dimensional structure according to the sample message that has detected; Focus by zoom microcobjective array (9), zoom each time, obtain a kind of spectrographic detection result of wave band, by repeating the focusing operation, can access the result of detection of the spectrum of different-waveband in this position, by the resulting image information of ccd video camera (15) after the contrast focusing each time, find out the most weak spectrum of light intensity that this position sensing arrives, then this spectrum is the color of this position of sample (10), detection image to each position of sample (10) all compares, and then can obtain the colouring information of sample; Photomultiplier PMT(16) the light intensity signal summation that will detect a depth direction on the sample is carried out opto-electronic conversion, by A/D converter (17) signal is transported to computing machine (18), spectrum with different frequency is carried out demodulation, carry out demodulation by frequency separately, modulation signal and the cosine signal with same carrier frequencies are multiplied each other, again by the unnecessary frequency spectrum of low-pass filter elimination, can obtain original signal, namely obtain the depth information of the sample that spectrum that this depth direction of sample namely has same frequency surveys.
2. according to the described color solid of claim 1 zone confocal microscopic imaging method, it is characterized in that, N í N formula H-PDLC grating array (4) is made up of N H-PDLC grating of N í (19) in the described step 1), each H-PDLC grating (19) is to be formed by polymkeric substance (20) and liquid crystal (21) alternate combinations, by being interfered, the PDLC material forms holographic grating between two glass plates that have an ITO film, H-PDLC grating (19) is placed with the grating fringe direction direction parallel with surface level, each H-PDLC grating (19) of white light continuous spectrum process is by light splitting, the spectrum of the different-waveband after the light splitting is separated from top to bottom successively, and the spectrum after the light splitting is through being focused onto the different depth position of sample (10) one directions behind the optical system device; Regulate driving circuit, light signal is modulated into square-wave signal, triangular signal, sinusoidal signal, to the copped wave of different-waveband spectrum, make the spectrum of same wave band have different frequencies, then through being focused onto the diverse location on (10) planes of sample behind the optical system device, H-PDLC grating array (4) carries out light splitting and chopping modulation to the white light continuous spectrum.
3. according to the described color solid of claim 2 zone confocal microscopic imaging method, it is characterized in that, described to setting different chopping frequencies respectively on N the H-PDLC grating of each N í (19), each frequency values satisfies the Nyquist sampling theorem, must not be higher than half of sample frequency, simultaneously, the carrier frequency of any two signals and their difference must be more than or equal to the twices of highest signal frequency.
4. according to the described color solid of claim 1 zone confocal microscopic imaging method, it is characterized in that, colour killing is loose microlens array (5) by can cancellation optical grating diffraction one-level forming with diffusing semi-permeable mirror (22) the N í N formula array of the colour killing of external spectrum in the described step 1), regulate the focus of each semi-permeable mirror (22) of the diffusing microlens array (5) of colour killing, each the H-PDLC grating (19) that is located at it corresponding H-PDLC grating array (4) is located, the light beam that light splitting modulates is by the diffusing microlens array (5) of colour killing, be adjusted into parallel beam and penetrate, form the parallel beam that different-waveband spectrum distributes from top to bottom successively in the space.
5. according to the described color solid of claim 1 zone confocal microscopic imaging method, it is characterized in that described step 1) and 2) in zoom microcobjective array (9) by with N í N formula H-PDLC grating array (4) in each H-PDLC grating (19) one by one corresponding magnification be that 40 times, numerical aperture are that 0.65 zoom microcobjective (23) is formed.
6. according to the described color solid of claim 1 zone confocal microscopic imaging method, it is characterized in that described step 2) in filter band (12) central small hole (26) diameter when equaling the Aili spot size, namely R=
, R is little pore radius, and D is the clear aperature of object lens (13), and F is the focal length of the diffusing object lens (11) of colour killing.
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