CN102608748B - Method for realizing multipath frequency division multiplexing fluorescent con-focal microscopic imaging by coaxial optical path - Google Patents
Method for realizing multipath frequency division multiplexing fluorescent con-focal microscopic imaging by coaxial optical path Download PDFInfo
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Abstract
The invention relates to a method for realizing multipath frequency division multiplexing fluorescent con-focal microscopic imaging by a coaxial optical path. The position of a focus is controlled by driving a polymer scattered liquid crystal coaxial lens array by a driving circuit, so as to realize chopping wave; a single-point control effect is achieved by controlling voltages applied to different liquid crystal boxes, and meanwhile, the method has the characteristics of high horizontal resolution, high longitudinal resolution, high time resolution, and the like; and a N*N type lens array is controlled, so that the multipoint parallel real-time detection can be realized. The method has various applications at the aspects of fluorescent con-focal detection and biology, such as, real-time observation for living cells, acquisition for information in the living cells and quantitative analysis for the acquired information. Compared with a present common con-focal microscopic detecting system, the method is more convenient and efficient.
Description
Technical field
The present invention relates to a kind of micro-imaging technique, particularly a kind of coaxial light path realizes multi-path frequency-division duplicating fluorescent confocal micro imaging method.
Background technology
The initial fundamental purpose of confocal microscopy is to eliminate the multiple scattered light that ordinary optical microscope produces when surveying sample.Laser scanning co-focusing microscope is a kind of advanced person's molecular biology and RESEARCH ON CELL-BIOLOGY instrument.It installs laser scanning device additional on the basis of fluorescent microscope imaging, in conjunction with datumization image processing techniques, gather fluorescence labeling image in tissue and cell, at subcellsular level, observe the variation of calcium plasma level, and in conjunction with technology such as electric physiology, observe the mutual relationship of cell physiological activity and cellular morphology and motion change.
The light that laser scanning co-focusing microscope sends for the focus place of focal plane of lens can well be assembled at pin hole place, can all by pin hole, be detected device and receive.And the light sending at focal plane upper-lower position can produce the hot spot that diameter is very large at pin hole place, the diameter of contrast pin hole, only has the light of few part to be detected device reception through pin hole.And along with the distance apart from focal plane of lens is larger, the parasitic light that sample produces is just larger at the disc of confusion at pin hole place, the energy that can see through pin hole is just fewer, thereby the signal producing on detector is just less, affects also less.Because its range of application is more extensive, become very important investigative technique in the research fields such as morphology, molecular cytobiology, Neuscience and pharmacology.
Confocal microscope has obtained huge development so far from producing, (sweep velocity is very fast from initial slit scan mode for scan mode, image resolution ratio is not high), to step scanning technology, (improved image resolution ratio, sample preparation requires too high), (sweep velocity is very fast to arrive drive-type optical beam scanner again, meet common focusing principle), the ultrawhite light that occur to use again photonic crystal fiber to produce is made the novel confocal microscopes such as the confocal Raman microscope of colored confocal microscope, 3-dimensional digital of exciting light source and the multiplexed confocal microscope of coupling fiber.The concept of frequency-division multiplexing fluorescent confocal micro-imaging and conception are born in 2006, at that time can only by Mach once moral light path and mechanical chopper realize double-path frequency-division multiplexing fluorescent confocal system, the volume of system is very large.In order really to realize multi-path frequency-division duplicating system, inventor once proposed to adopt and realized multi-path frequency-division duplicating fluorescent confocal imaging system from axle holographic lens array, but due to very high from axle system angle requirement, the trouble that has caused system to realize.For this reason, we have proposed again to adopt the hyperchannel frequency-division multiplexing fluorescent confocal implementation method of coaxial electric control zooming Polymer Dispersed Liquid Crystal.The Primary Component of this system is coaxial liquid crystal lens, has integrated form design, and easy operating is realized confocal and out of focus by face type and electric field regulation and control and controlled.Make so the concrete realization of concept of frequency-division multiplexing fluorescent confocal become easy operation, be easy to the system that realizes, to have volume little for this system simultaneously, the advantages such as movement-less part.
Summary of the invention
The problem that the present invention be directed to existing traditional confocal microscope and exist from axle multi-path frequency-division duplicating fluorescent confocal microscopic system, propose a kind of coaxial light path and realized multi-path frequency-division duplicating fluorescent confocal micro imaging method, system adopts N * N formula lens arra to realize copped wave, can realize the parallel real time high-speed of multiple spot fluorescence signal and survey, and there is the features such as high spatial resolution and temporal resolution.
Technical scheme of the present invention is: a kind of coaxial light path realizes multi-path frequency-division duplicating fluorescent confocal micro imaging method, specifically comprises the steps:
1) build N * N passage multifrequency modulated laser light path: laser instrument, beam expanding lens, coaxial H-PDLC lens arra, filter band, lens are coaxially built successively, and remain level with platform, the light beam coupling that the wavelength that 30mw collimation semiconductor laser sends is 405nm is entered beam expanding lens, the light beam of beam expanding lens outgoing arrives the chopping modulation of carrying out light splitting and different frequency on the coaxial H-PDLC lens arra of N * N formula, makes the focus of the laser by the liquid crystal cell array on the coaxial H-PDLC lens arra of N * N formula be in the aperture position on filter band;
2) build the microscopical fluorescence excitation light path of N * N road frequency-division multiplexing fluorescent confocal: by focal plane also at the lens of the focal plane F of coaxial H-PDLC lens arra, the N of the focusing through the outgoing of H-PDLC lens arra * N bundle light is adjusted into parallel beam to be penetrated, be coupled in dichroic mirror group, it is 40 times that the laser of the 405nm exciting through dichroic mirror group is injected into enlargement ratio by coupling, numerical aperture is 0.65 infinity oil immersion microcobjective, biological sample is put on three-dimensional trim holder, adjusting three-dimensional trim holder makes biological sample just on microcobjective focal plane, light beam will be focused to n under the effect of object lens on biological sample
2individual luminous point, inspires fluorescence,
3) build n
2the microscopical micro-imaging of road frequency-division multiplexing fluorescent confocal: the 520nm-540nm green fluorescence by biological sample surface excitation will be with exciting light along same straight line under sample and the orthogonal condition of microcobjective, oppositely pass through microcobjective, become parallel beam, incide in dichroic mirror group, fluorescence is all gone out from the transmission of dichroic mirror group, the fluorescence incident Amici prism transmiting carries out light splitting for the second time, be divided into two parts, wherein a part is gathered ccd video camera by achromat and finally in computing machine, in software, is obtained sample cell image by the signal of CCD collected specimens, another part fluorescence is transported in photomultiplier PMT by fibre coupler arrays coupling merga pass optical fiber through lens, this signal belongs to cofocus scanning microscopic signal,
4) realization of signals collecting and processing section: photomultiplier PMT is by according to exciting the n obtaining in biological sample
2the size of the fluorescence intensity at individual phosphor dot place, by the picture signal at phosphor dot place, pass through opto-electronic conversion, formal output with electric signal, by the output termination voltage amplifier circuit of photomultiplier, the feeble signal that voltage amplifier circuit collects PMT is carried out voltage amplification, access data capture card, finally by USB interface, signal data is sent into computing machine, computing machine is by Matlab software, coding, to modulate fluorescence signal by wave filter elimination higher hamonic wave and part noise, and the fluorescence signal through ovennodulation is separated, by modulating frequency separately, undertaken obtaining original fluorescence signal after demodulation, obtain required sample message.
Described coaxial H-PDLC lens arra is by N * N liquid crystal cell, the input lead of liquid crystal cell, and the para tape between each liquid crystal cell forms.Described liquid crystal cell comprises four parts: dome-type glass cover, fixed polar plate, dead ring, plain conductor, first dead ring and dome-type glass cover are combined, between them, insert a plain conductor, the ITO film of confirming plain conductor and dome-type glass cover inside surface intersects, the wire of drawing is extracted and is applied voltage by para tape, H-PDLC material reinjects, again fixed polar plate and above-mentioned integral body are assembled up, on fixed polar plate, scribble ITO film, area and dead ring inner ring surface are long-pending to be equated, for making alive convenience, ITO film on fixed polar plate is extended out, wire on para tape is connected with two electrodes of single liquid crystal cell, two electrodes are on plain conductor and fixed polar plate, to scribble ITO film.
The lateral resolution of confocal microscope in described step 4)
and axial resolution
be expressed as:
,
, wherein,
for inspired glimmering light wavelength,
the numerical aperture that represents microcobjective,
by the effective refractive index of detection sample, in sample, 70% is water, can setting value be 1.3.
In described step 4), the detailed process of demodulation is, first modulation signal and the cosine signal with same carrier frequencies is multiplied each other, then by the unnecessary frequency spectrum of low-pass filter elimination, can obtain original signal.
Beneficial effect of the present invention is: the coaxial light path of the present invention realizes multi-path frequency-division duplicating fluorescent confocal micro imaging method, owing to driving the coaxial lens arra of Polymer Dispersed Liquid Crystal by driving circuit, control the position of focus, thereby realize copped wave, by control, be applied to the voltage on different liquid crystal cells, can reach the effect that single-point is controlled, also there is lateral resolution, longitudinal frame, temporal resolution high simultaneously, due to what control, be N * N formula lens arra, can realize the parallel real-time detection of multiple spot.Fluorescent confocal survey and biological aspect have many application, as carried out real-time monitored to active somatic cell, obtain the information in living cells, and obtained information carried out to quantitative test, more convenient, efficient than current general confocal micro detection system.
Accompanying drawing explanation
Fig. 1 is the microscopical light path schematic diagram of N * N of the present invention road frequency-division multiplexing fluorescent confocal;
Fig. 2 is the coaxial H-PDLC lens arra of N * N formula of the present invention schematic diagram;
Fig. 3 is the single liquid crystal cell schematic perspective view of the present invention;
Fig. 4 is the single dome-type glass cover of the present invention sectional view;
Fig. 5 is the dead ring schematic perspective view that the single liquid crystal cell of the present invention is corresponding;
Fig. 6 is the fixed polar plate schematic diagram that the single liquid crystal cell of the present invention is corresponding;
Fig. 7 is the light focal variation schematic diagram of lens of the present invention scioptics when powering up and not powering up;
Fig. 8 is array of orifices filter band schematic diagram of the present invention.
Embodiment
It is as follows that coaxial light path realizes multi-path frequency-division duplicating fluorescent confocal micro imaging method performing step:
A: build N * N passage multifrequency modulated laser light path: light path schematic diagram as shown in Figure 1, laser instrument 1, beam expanding lens 2, coaxial H-PDLC lens arra 3, filter band 4, lens 5 are built successively.In this process, should be noted light beam should be as far as possible from the center of each device by and remain level with platform.First the light beam coupling that is 405nm by the wavelength being sent by 30mw collimation semiconductor laser 1 is entered beam expanding lens 2(and which kind of long wavelength laser to be depended on the fluorescence labels in biological sample with, biological specimen is higher to the susceptibility of royal purple light in this experiment, can inspire wavelength coverage at the green fluorescence of 520nm-540nm).Make the light beam of beam expanding lens 2 outgoing arrive the chopping modulation of carrying out light splitting and different frequency on the coaxial H-PDLC lens arra 3 of N * N formula, regulation voltage, make the focus of the laser by liquid crystal cell array be in the aperture position on filter band 4, aperture size more approaches ideal point, resolution is higher, and image quality is better.The theoretical proof of calculating can guarantee high resolving power and the chromatography ability of confocal imaging when the diameter of aperture equals Aili spot size, has again enough luminous energy to be detected device by aperture and receives.Regulate lens arra, determine that the focal plane of lens arra is on the F of position.The coaxial H-PDLC lens arra of N * N formula schematic diagram as shown in Figure 2, wherein 18 is liquid crystal cell, specification is that overall diameter is 5mm, interior diameter is 3mm, 19 is wire, 20 is para tape, width is 4mm, the chopper effect of coaxial H-PDLC lens arra 3 is to drive the dome-type liquid crystal cell 18 of having filled H-PDLC material by driving circuit, namely utilize the displacement of liquid crystal under voltage drives, make the variations in refractive index of liquid crystal cell, thereby make to change through the light focal position of liquid crystal cell, by the filter action of aperture, realize copped wave.Can regulate driving circuit is square-wave signal by the optical signal modulation of continuous strength, sine wave signal, triangular signal or other shape waveform signals.Conventionally by the optical signal modulation of continuous strength, be square-wave signal.In this experiment, through the luminous energy that excites of ovennodulation, from the fluorescence labels of biological sample 8, inspire the fluorescence through same carrier frequency modulation, thereby make fluorescence signal there is certain frequecy characteristic, n
2individual copped wave passage is set respectively certain carrier frequency value.Each frequency values all should meet Nyquist sampling theorem, must not be higher than half of sample frequency, and as described above must not be higher than 125KHz.Meanwhile, the twice that the carrier frequency of any two signals and their difference must be more than or equal to highest signal frequency can solution tune to open to guarantee the two paths of signals of demodulation.
B: build the microscopical fluorescence excitation part of N * N road frequency-division multiplexing fluorescent confocal light path: by focal plane also at the lens 5 of the focal plane F of coaxial H-PDLC lens arra 3, the N of the focusing from lens arra outgoing * N bundle light is adjusted into parallel beam to be penetrated, be coupled in dichroic mirror group 6, it is the infinity oil immersion microcobjective 7 that 40 times, numerical aperture are 0.65 that the laser of the 405nm exciting through dichroic mirror group 6 is injected enlargement ratio by coupling.In coupling process, should be noted that and the direction of propagation of light beam is adjusted as far as possible consistent with the central axis direction of microcobjective, to guarantee excitation beam vertical incidence microcobjective 7.Biological sample 8 is put on three-dimensional trim holder, adjusted the knob of three-dimensional trim holder to change the distance of 7 of biological sample 8 and microcobjectives, make sample 8 just on microcobjective 7 focal planes.Light beam will be focused to n under the effect of object lens on biological sample 8
2individual luminous point, inspires fluorescence.Now, through coaxial H-PDLC lens focus, at the 405nm of focal plane laser point light source and biological specimen, just formed a pair of confocal relation.
C: build n
2the microscopical micro-imaging of road frequency-division multiplexing fluorescent confocal: the 520nm-540nm green fluorescence by biological sample 8 surface excitations will be with exciting light along same straight line under sample and the orthogonal condition of microcobjective 7, oppositely by microcobjective 7, become parallel beam, get to dichroic mirror group 6.Fluorescence is all gone out from 6 transmissions of dichroic mirror group.This bundle fluorescence incident Amici prism 9 is carried out to light splitting for the second time, obtain the identical two parts of each property such as intensity.Wherein a part is gathered ccd video camera 11 by achromat 10 and finally in computing machine, in software, is obtained sample cell image by the signal of CCD collected specimens.Another part fluorescence is transported in photomultiplier PMT15 by fibre coupler arrays 13 coupling merga pass optical fiber 14 through lens 12, and this signal belongs to cofocus scanning microscopic signal.The lateral resolution of this confocal microscope
and axial resolution
be expressed as:
,
, wherein,
for inspired glimmering light wavelength,
the numerical aperture that represents microcobjective,
by the effective refractive index of detection sample, in sample, 70% is water, can setting value be 1.3.Suppose that the wavelength of fluorescence inspiring is 530nm, the numerical aperture of microcobjective
be 0.65, lateral resolution
be 0.5 μ m, axial resolution
it is 0.55 μ m left and right.Temporal resolution is subject to the sample frequency of data collecting card and the restriction of selected chopping frequency.
D: making and characteristic based on the coaxial lens arra chopping the light modulation of Polymer Dispersed Liquid Crystal: as the critical component of confocal chopping modulation, the coaxial H-PDLC lens arra 3 of N * N formula can be realized light splitting simultaneously, focus on frequency modulation function.Single liquid crystal cell schematic perspective view as shown in Figure 3, this liquid crystal cell is comprised of four parts: dome-type glass cover 22, fixed polar plate 25, dead ring 23, wire 24.The object that adopts lens focus is herein exactly to make light source and the focal plane of placing the infinity microcobjective of sample meet conjugate relation, realize confocal, so just can elimination afocal spurious signal, to improve entire system detectivity.By power control system, can to light beam, modulate different chopping frequencies respectively simultaneously, realize frequency modulation function.In liquid crystal cell 18, dead ring 23 and dome-type glass cover 22 combine, and insert therein a plain conductor 24, confirm that the ITO film 26 of plain conductor 24 and dome-type glass cover inside surface intersects.Inject H-PDLC material 21, single semisphere glass cover sectional view is as Fig. 4.As shown in Figure 5, overall diameter is that 5mm, interior diameter are 3mm, thick 0.5mm to dead ring 23 corresponding to the single liquid crystal cell of the present invention.Fig. 6 is the fixed polar plate schematic diagram that the single liquid crystal cell of the present invention is corresponding, treat that dome-type glass cover 22 and dead ring 23 assemble, and insert therein a plain conductor 24, guarantee that plain conductor 24 and dome-type glass cover 22 inside surface ITO films 26 intersect and inject after H-PDLC, fixed polar plate and above-mentioned integral body are assembled up, on fixed polar plate, scribble ITO film 27, its area is that dead ring 23 inner ring surfaces are long-pending, for making alive convenience, ITO film 27 on fixed polar plate is extended out (development length is relevant with the particular location of single liquid crystal cell), and draw with wire.Each fixed polar plate is separated by para tape 20, the wire 19 on para tape 20 be connected with two electrodes of single liquid crystal cell (is the plain conductor 24 between dead ring and dome-type glass cover, and another is the ITO film 27 on fixed polar plate).Change voltage voltage and cause the focus of liquid crystal cell array to change, as shown in Figure 7.Wherein, after making alive, focus, on the aperture of filter band, removes voltage, and focus, not on the aperture of filter band, according to this characteristic, just can be carried out copped wave with this liquid crystal cell array.What Fig. 8 showed is the structure of array of orifices wave filter, and little pore size is
.Wherein,
for irradiating optical wavelength,
for liquid crystal cell focal length now,
diameter for liquid crystal cell.
E: the realization of signals collecting and processing section: photomultiplier PMT15 is by according to exciting the n obtaining in biological sample
2the size of the fluorescence intensity at individual phosphor dot place, by the picture signal at phosphor dot place, by opto-electronic conversion, with the formal output of electric signal.By the output termination voltage amplifier circuit 16 of photomultiplier.Voltage amplifier circuit is comprised of LH05-10A05 AC/DC converter and the low offset voltage dynamic amplifier of OP07, and the feeble signal that its role is to PMT to collect is carried out voltage amplification.Subsequently, then by the output access data capture card of voltage amplifier circuit 16, finally by USB interface, signal data is sent into computing machine 17.By Matlab software, coding, will modulate fluorescence signal by wave filter elimination higher hamonic wave and part noise, and the fluorescence signal through ovennodulation will be separated.By modulating frequency separately, undertaken obtaining original fluorescence signal after demodulation, obtain required sample message.The detailed process of demodulation is first modulation signal and the cosine signal with same carrier frequencies to be multiplied each other, then by the unnecessary frequency spectrum of low-pass filter elimination, can obtain original signal.
Frequency division multiplexing multichannel fluorescent confocal microscopic imaging system consists of multi-path frequency-division duplicating fluorescence confocal microscope and collection signal disposal system.
Multi-path frequency-division duplicating fluorescence confocal microscope is the confocal microscope that combines frequency multiplexing technique.The present invention adopts N * N formula lens arra, is characterized in accomplishing n
2the point of individual diverse location scans simultaneously and is processed into picture, more quick, efficient, also has higher lateral resolution, longitudinal frame and signal to noise ratio (S/N ratio) simultaneously, also has the higher features such as temporal resolution.
This patent has built the n of N * N array
2road frequency-division multiplexing fluorescent confocal microscopic imaging system.The main composition of system comprises: what excitation source adopted is the 30mw collimation semiconductor laser (Shanghai dream laser Science and Technology Ltd. product) of outgoing 405nm bluish violet light beam, the chopping the light frequency modulation device of Polymer Dispersed Liquid Crystal lens arra N * N, the lens arra of this H-PDLC adopts the clear glass of the dome-type of Polymer Dispersed Liquid Crystal filling to make, (enlargement ratio is 40X to infinity microcobjective, numerical aperture is 0.65), the purple wave band dichroic mirror group (Shanghai rectangular optical instrument company limited provides) of the separated 405nm royal purple light of energy and 520-540nm green glow, CR186 type fast phtotmultiplier tube (is called for short PMT, response times 4 μ s, spectral response range is 300nm～650nm, peak wavelength is 420nm, anode gain is 1.88 * 106, shore, Beijing pine photon provides) and high-resolution color ccd video camera (highest resolution 1280 * 1024 pixels, the DH-M1300UC that Daheng's image provides).
In addition, also comprise optical fiber and fiber coupler group, photomultiplier (PMT) and voltage amplifier circuit and data collecting card etc., complete respectively that fluorescence signal is confocal, signals collecting, signal amplification and the function such as modulus, digital-to-analog conversion.
As shown in Figure 1, first laser beam is divided into n by beam expanding lens 2 by H-PDLC lens arra 3 to constructed N * N passage confocal micro imaging system structure
2individual part, with driving voltage, by 3 couples of every Shu Guang of coaxial H-PDLC lens arra, carry out the modulation of different frequency, then through confocal lens system, become parallel beam, parallel beam is by dichroic mirror group 6DM(through dichroic mirror group DM, and the exciting light of the royal purple wave band of 405nm and green fluorescence signal all have unidirectional transport property).The n of 405nm
2bundle exciting light focuses on respectively the n of biological sample 8
2individual diverse location, and produced the fluorescence that wavelength is 520nm-540nm; This n
2bundle fluorescence passes through microcobjective optically focused subsequently, the veiling glare by 6 eliminations of dichroic mirror group except green fluorescence, then enter Amici prism 9BS, be divided into the same two parts of intensity phase of equality.Wherein a part, by achromatism image-forming objective lens 10, images in ccd video camera.And after 12 focusing of another part fluorescence line focus lens, through prism by the fluorescence signal of every passage separately, then be coupled to the rear input photomultiplier of optical fiber 14 PMT15 by fiber coupler group 13.
For the realization of the chopper of the coaxial H-PDLC lens arra 3 in fluorescence confocal microscope light path, be by realizing the control of coaxial H-PDLC lens arra 3 automatically controlled frequency modulation, its n
2the frequency modulating signal on road must meet certain requirement.First, for meeting Nyquist sampling theorem, the sample frequency of data collecting card must be more than or equal to the twice of modulating frequency, and in experimental system, data collecting card sample frequency is 250KHz, should be less than or equal to 125KHz.Secondly, for preventing that the frequency of each road light signal from occurring that overlapping phenomenon causes each road signal mutually to be crosstalked, the carrier frequency of two adjacent signals and their difference must be more than or equal to the twice of the highest fluorescence signal change frequency.
In signal collection processing section, in experimental system, voltage amplifier circuit 16 is comprised of LH05-10A05 AC/DC converter and the low offset voltage dynamic amplifier of OP07, and the faint data that its role is to PMT to collect are carried out voltage amplification.Subsequently, adopt USB2816 type data collecting card (sample frequency is 250KHz, and Beijing Altay company provides) that the output signal of amplifying circuit is sent into computing machine by USB interface.By Matlab software, modulation fluorescence signal is carried out obtaining original fluorescence signal after filtering and demodulation again, obtain each phosphor dot fluorescence intensity temporal evolution information.
Claims (2)
1. coaxial light path realizes a multi-path frequency-division duplicating fluorescent confocal micro imaging method, it is characterized in that, specifically comprises the steps:
1) build N * N passage multifrequency modulated laser light path: by laser instrument (1), beam expanding lens (2), coaxial H-PDLC lens arra (3), filter band (4), lens (5) are coaxially built successively, and remain level with platform, the light beam coupling that the wavelength that 30mw collimation semiconductor laser (1) sends is 405nm is entered beam expanding lens (2), the light beam of beam expanding lens (2) outgoing arrives the chopping modulation of carrying out light splitting and different frequency on N * N coaxial H-PDLC lens arra of formula (3), make the focus of the laser by the liquid crystal cell array on the coaxial H-PDLC lens arra of N * N formula (3) be in the aperture position on filter band (4),
2) build the microscopical fluorescence excitation light path of N * N road frequency-division multiplexing fluorescent confocal: by focal plane also at the lens (5) of the focal plane F of coaxial H-PDLC lens arra (3), the N of the focusing through H-PDLC lens arra (3) outgoing * N bundle light is adjusted into parallel beam to be penetrated, be coupled in dichroic mirror group (6), it is 40 times that the laser of the 405nm exciting through dichroic mirror group (6) is injected into enlargement ratio by coupling, numerical aperture is 0.65 infinity oil immersion microcobjective (7), biological sample (8) is put on three-dimensional trim holder, adjusting three-dimensional trim holder makes biological sample (8) just on microcobjective (7) focal plane, light beam will be focused to n under the effect of object lens on biological sample (8)
2individual luminous point, inspires fluorescence,
3) build n
2the microscopical micro-imaging of road frequency-division multiplexing fluorescent confocal: the 520nm-540nm green fluorescence by biological sample (8) surface excitation will be with exciting light along same straight line under sample and the orthogonal condition of microcobjective (7), oppositely by microcobjective (7), become parallel beam, incide in dichroic mirror group (6), fluorescence is all gone out from dichroic mirror group (6) transmission, the fluorescence incident Amici prism (9) transmiting carries out light splitting for the second time, be divided into two parts, wherein a part converges to ccd video camera (11) by achromat (10) and finally in computer software, obtains sample cell image by the signal of CCD collected specimens, another part fluorescence is transported in photomultiplier PMT (15) by fibre coupler arrays (13) coupling merga pass optical fiber (14) through lens (12), this signal belongs to cofocus scanning microscopic signal,
4) realization of signals collecting and processing section: photomultiplier PMT (15) is by according to exciting the n obtaining in biological sample
2the size of the fluorescence intensity at individual phosphor dot place, by the picture signal at phosphor dot place, pass through opto-electronic conversion, formal output with electric signal, by the output termination voltage amplifier circuit (16) of photomultiplier, the feeble signal that voltage amplifier circuit (16) collects PMT (15) is carried out voltage amplification, access data capture card, finally by USB interface, signal data is sent into computing machine (17), computing machine (17) is by Matlab software, coding, to modulate fluorescence signal by wave filter elimination higher hamonic wave and part noise, and the fluorescence signal through ovennodulation is separated, by modulating frequency separately, undertaken obtaining original fluorescence signal after demodulation, obtain required sample message.
2. coaxial light path realizes multi-path frequency-division duplicating fluorescent confocal micro imaging method according to claim 1, it is characterized in that, described coaxial H-PDLC lens arra (3) is by N * N liquid crystal cell (18), the input lead (19) of liquid crystal cell (18), para tape (20) between each liquid crystal cell (18) forms, described liquid crystal cell (18) comprises four parts: dome-type glass cover (22), fixed polar plate (25), dead ring (23), plain conductor (24), first dead ring (23) and dome-type glass cover (22) are combined, between them, insert a plain conductor (24), the ITO film (26) of confirming plain conductor (24) and dome-type glass cover inside surface intersects, the wire of drawing is extracted and is applied voltage by para tape, H-PDLC material (21) reinjects, again fixed polar plate (25) and above-mentioned integral body are assembled up, on fixed polar plate (25), scribble ITO film (27), area and dead ring (23) inner ring surface is long-pending to be equated, for making alive convenience, ITO film (27) on fixed polar plate (25) is extended out, wire (19) on para tape (20) is connected with two electrodes of single liquid crystal cell, two electrodes are to scribble ITO film (27) on plain conductor (24) and fixed polar plate (25).
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| KR100850022B1 (en) * | 2007-11-28 | 2008-08-04 | 부산대학교 산학협력단 | Transreflective hologram display using polyurethane matrix |
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