CN109799602A - A kind of light microscopic imaging device and method based on line scanning space-time focusing - Google Patents
A kind of light microscopic imaging device and method based on line scanning space-time focusing Download PDFInfo
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Abstract
The embodiment of the present invention provides a kind of light microscopic imaging device and method based on line scanning space-time focusing, which includes: that laser emitting module generates nonlinear excitation pulse light；Line scan module obtains the angle dispersion and deflection angle of excitation pulse light, and the excitation pulse light is scanned sample；Optical microphotograph module forms the focal line focused simultaneously in two dimensions in space and time in the sample, to generate transmitting optical signal based on nonlinear optical effect excitation sample；Synchronous filtering module detects the transmitting optical signal of sample, and is filtered to transmitting optical signal；It rebuilds module and 2-d reconstruction is carried out according to the transmitting optical signal that synchronous filtering module detects, obtain the reconstructed image of sample.The present invention can effectively inhibit signal cross-talk caused by stray light, have many advantages, such as wide visual field, high spatial resolution, high time resolution, deep penetration depth, can provide abundant information for biological dynamic process research, medical diagnosis on disease foundation etc..
Description
Technical field
The present embodiments relate to optical technical field more particularly to it is a kind of based on line scanning space-time focus light it is micro- at
As device and method.
Background technique
Optical microscopy imaging has many advantages, such as that non-intrusion type, high sensitivity, high-resolution, imaging scale are flexible, it has also become
The technological means of biomedical research indispensability.Currently, optical microscopy imaging has been not only applicable to biodynamic process study, also answer
For numerous areas such as clinical disease diagnosis.But since biological tissue has anisotropy and heterogeneity, light is at biological group
Random refraction and scattering will be undergone when knitting middle propagation, be caused signal cross-talk, be reduced the signal-to-noise ratio of imaging.
In order to overcome the influence of biological tissue scatters, people have developed laser scanning confocal microtechnic, i.e., by
End of probe introduces confocal pinhole and filters out stray light caused by scattering, to improve the signal-to-noise ratio of imaging.But due to the technology
It is to be based on Single Photon Absorption effect, will all inspires fluorescence in entire excitation light path, and cause to cannot be neglected phototoxicity.Multi-photon
The appearance of microtechnic, dexterously solves the above problem.So-called multi-photon microscopy, refers to based on nonlinear optical effect
The method for generating optical signal.
Different from Single Photon Absorption effect, nonlinear optical effect needs the multiple photons of transient absorption that could occur, and produces
Raw probability is very low, usually only generates at focusing focus, to realize local excition.Further, since nonlinear optical effect
It needs to excite using longer wavelength, this not only lowers phototoxicities, can also be effective against scattering, improve imaging depth.
Conventional multi-photon microscopy mostly uses greatly spot scan mode to be imaged point by point, due to the machinery of scanning element
Inertia, there is 'bottleneck' restrictions for image taking speed.In order to improve image taking speed, the concept of wide field imaging can be used for reference, therefore go out
Space-time focusing technology is showed, i.e., excitation light pulse has been focused by time domain, two, airspace dimension, has realized that wide field chromatography swashs
Hair.
According to the shape of excitation area, space-time confocal microscopy, which can be divided into line, to be swept space-time and focuses micro- and face and excite space-time
It focuses micro-.Wherein, the former realizes bar shaped (line) excitation on object lens focal plane, therefore need to only be swept in perpendicular direction
Two-dimensional imaging can be carried out by retouching, thus referred to as line sweep space-time focus it is micro-.Although being pointed out that space-time confocal microscopy base
The wide field chromatography excitation of deep tissues is realized in nonlinear optical effect, but its excited optical signals will be organized
The influence of scattering causes signal cross-talk, therefore imaging depth will be limited when being detected using wide field.
Summary of the invention
In view of the above-mentioned problems, the embodiment of the present invention provide it is a kind of based on line scanning space-time focus light microscopic imaging device and
Method.
In a first aspect, the embodiment of the present invention provides a kind of light microscopic imaging device focused based on line scanning space-time, comprising:
Laser emitting module, for generating nonlinear excitation pulse light；
Line scan module for obtaining the angle dispersion and deflection angle of the excitation pulse light, and makes the excitation pulse
Light is scanned sample；
Optical microphotograph module, for forming the focusing focused simultaneously in two dimensions in space and time in the sample
Line, to excite the sample to generate transmitting optical signal based on nonlinear optical effect；
Synchronous filtering module is filtered for detecting the transmitting optical signal of the sample, and to the transmitting optical signal,
The trigger signal of the synchronous filtering module is synchronous with the trigger signal of the line scan module；
Module is rebuild, the transmitting optical signal for detecting according to the synchronous filtering module carries out 2-d reconstruction, obtains
The reconstructed image of the sample.
Second aspect, the embodiment of the present invention provide a kind of light micro imaging method focused based on line scanning space-time, comprising:
Generate nonlinear excitation pulse light；
The angle dispersion and deflection angle of the excitation pulse light are obtained, and the excitation pulse light sweeps sample
It retouches；
The focal line focused simultaneously in two dimensions in space and time is formed, in the sample to be based on nonlinear optics
Effect excites the sample to generate transmitting optical signal；
The transmitting optical signal of the sample is detected, and the transmitting optical signal is filtered, the synchronous filtering module
Trigger signal it is synchronous with the trigger signal of the line scan module；
2-d reconstruction is carried out according to the transmitting optical signal that the synchronous filtering module detects, obtains the reconstruct of the sample
Image.
A kind of light microscopic imaging device focused based on line scanning space-time provided in an embodiment of the present invention and method, can be effective
Ground inhibits signal cross-talk caused by stray light, has wide visual field, high spatial resolution, high time resolution, deep penetration depth etc.
Advantage can provide abundant information for biological dynamic process research, medical diagnosis on disease foundation etc..
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is this hair
Bright some embodiments for those of ordinary skill in the art without creative efforts, can be with root
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is a kind of structural schematic diagram that the light microscopic imaging device that space-time focuses is scanned based on line of the embodiment of the present invention；
Fig. 2 is a kind of schematic diagram that the light microscopic imaging device that space-time focuses is scanned based on line of the embodiment of the present invention；
Fig. 3 is a kind of structural representation that the light microscopic imaging device that space-time focuses is scanned based on line of one embodiment of the invention
Figure；
Fig. 4 is a kind of structural representation that the light microscopic imaging device that space-time focuses is scanned based on line of further embodiment of this invention
Figure；
Fig. 5 is a kind of flow chart that the light micro imaging method that space-time focuses is scanned based on line of the embodiment of the present invention.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
Fig. 1 is a kind of structural schematic diagram that the light microscopic imaging device that space-time focuses is scanned based on line of the embodiment of the present invention,
As shown in Figure 1, the device includes: laser emitting module 101, line scan module 102, optical microphotograph module 103, synchronous filtering mould
Block 104 and reconstruction module 105, in which:
Laser emitting module 101, for generating nonlinear excitation pulse light；
Line scan module 102 for obtaining the angle dispersion and deflection angle of the excitation pulse light, and makes the excitation arteries and veins
It washes off and sample is scanned；
Optical microphotograph module 103, for being formed in the sample simultaneously in the poly- of the two dimensions focusing in space and time
Focal line, to excite the sample to generate transmitting optical signal based on nonlinear optical effect；
Synchronous filtering module 104 is filtered for detecting the transmitting optical signal of the sample, and to the transmitting optical signal
Wave, the trigger signal of the synchronous filtering module are synchronous with the trigger signal of the line scan module；
Module 105 is rebuild, the transmitting optical signal for detecting according to the synchronous filtering module carries out 2-d reconstruction, obtains
Take the reconstructed image of the sample.
The excitation pulse light of 101 parametrical nonlinearity of laser emitting module, line scan module 102 are the lines focused based on space-time
Scanning system, obtains the angle dispersion and deflection angle of excitation pulse light, and excitation pulse light is scanned sample, and optics is aobvious
Micromodule 103 forms the focal line focused simultaneously in two dimensions in space and time in the sample, to be imitated based on nonlinear optics
Stress send out sample generate transmitting optical signal, synchronous filtering module 104 detect sample transmitting optical signal, and to transmitting optical signal into
Row filtering, the trigger signal of synchronous filtering module is synchronous with the trigger signal of the line scan module, rebuilds module 105 according to same
The transmitting optical signal that step filter module detects carries out 2-d reconstruction, obtains the reconstructed image of sample.
A kind of light microscopic imaging device focused based on line scanning space-time provided in an embodiment of the present invention, can effectively inhibit
Signal cross-talk caused by stray light has many advantages, such as wide visual field, high spatial resolution, high time resolution, deep penetration depth, can
Abundant information is provided for biological dynamic process research, medical diagnosis on disease foundation etc..
On the basis of the above embodiments, it is preferable that further include optical beam transformation module, the optical beam transformation module is located at institute
Before stating line scan module, the optical beam transformation module is used to adjust the beam sizes of the excitation pulse light.
The light microscopic imaging device further includes optical beam transformation module, and optical beam transformation module is located at after laser emitting module,
Before line scan module, optical beam transformation module is used to modulate the beam sizes of excitation pulse light.
On the basis of the above embodiments, it is preferable that the line scan module includes diffractive-optical element, lens and optics
Scanning element, the diffractive-optical element are located at the object space focal plane of the lens, and the optical scanning element is located at the lens
Image space focal plane, the diffractive-optical element is used to obtain the angle dispersion of excitation pulse light adjusted, the optical scanner member
Part is used to obtain the variable deflection angle of excitation pulse light adjusted.
Specifically, line scan module is placed in after optical beam transformation module, specifically includes diffractive-optical element, lens and optics
Scanning element, diffractive-optical element and optical scanning element are respectively placed in the object space focal plane and image space focal plane of lens；Optical diffraction
Element is used to introduce the angle dispersion of excitation pulse light beam, and optical scanning element is used to introduce the variable deflection of excitation pulse light beam
Angle；
Optical microphotograph module is placed in after above-mentioned optical scanning element, including lens group and microcobjective, is connected by lens group
Entrance pupil face after above-mentioned optical scanning element and microcobjective is connect, and constitutes 4f system, for being formed simultaneously in space in the sample
And the focal line that two dimensions of time focus, to excite tissue sample and generate transmitting optical signal based on nonlinear optical effect；
Synchronous filtering module is placed in transmitting optical signal in the linear scanning system focused based on space-time and collected simultaneously through microcobjective
After the optical scanning element of reverse transfer, including filter plate, two-dimensional detector, the transmitting optical signal for selecting sample are gone forward side by side
Traveling optical signal detection；Wherein, the acquisition of information of two-dimensional detector and the trigger signal of the linear scanning system focused based on space-time are same
Step.
It rebuilds the information that module is detected two-dimensional detector and carries out calculating reconstruction, recover high-quality signal.
Further, laser emitting module is ultra-short pulse laser light source, is additionally provided with before ultra-short pulse laser output
Dispersion pre-compensation system, the dispersion accumulated before reaching microcobjective focusing surface for pre-compensating for ultrashort pulse.
Further, the linear scanning system based on space-time focusing technology further includes being placed in diffractive-optical element to pass along exciting light
It broadcasts adaptive optical element of the direction at the Fourier plane after lens and further overcomes life for carrying out spectrum phase shaping
Influence of the object sample scattering to chromatography excitation quality.
In ultra-short pulse laser light source and optical beam transformation module, ultra-short pulse laser light source, can according to output pulse width
Select femtosecond pulse light source or picosecond pulse laser light source.
Whether ultra-short pulse laser light source is adjustable according to output wavelength, and the ultra-short pulse laser light source of fixed wave length can be selected
Or the ultra-short pulse laser light source of tunable wavelength.
Optical beam transformation module is Galilean telescope system or Kepler telescope system.
Ultra-short pulse laser light source and optical beam transformation module provide non-linear in the exciting light for generating non linear optical signal
Optical signalling passes through two-photon absorption fluorescent effect, Three-photon absorption fluorescent effect, two-photon excitation-fluorescence resonance energy transfer
Effect, second_harmonic generation effect, triple-frequency harmonics generate effect, stimulated raman scattering or coherent anti-Stokes Raman and dissipate
Penetrate the generation of any one of effect.
In line scan module, grating, distorting lens, spatial light modulator or other optical diffractions are can be selected in diffractive-optical element
Element；Optical scanning element selects galvanometer, polygonal mirror or acousto-optic modulator etc..
In synchronous filtering module, filter plate selects dichroic mirror, band pass filter, low pass filters or long pass filter piece.
Two-dimensional area detector be placed in biological sample excitation face conjugate planes, focal line vertical direction width by sample
The design size of conjugate image determines.
Two-dimensional area detector selects charge coupled cell (CCD), electron multiplying charge coupling element (EMCCD) or Scientific Grade
Complementary mos device (sCMOS) etc..
Fig. 2 is a kind of schematic diagram that the light microscopic imaging device that space-time focuses is scanned based on line of the embodiment of the present invention, Fig. 2 a
It is shown the stimulation effect figure that line sweeps space-time focusing, wherein the horizontal line of top half is focal line, and base map is neuronal cell
Schematic diagram.Fig. 2 b is distributed original signal, is scattered rear signal distributions and uses conventional detection scheme, expansion proposed by the invention
Exhibition detection and calculating, which are rebuild, to be illustrated.
It is specific as follows: using nonlinear optical effect and to combine space-time focusing technology (U.S. patent Nos
US20080151238A1 the focal line (direction x) with high axial resolution, the light excited) can be generated on biological sample
Signal is in 2 D photoelectric test surface (Sx,Sy) on be imaged, wherein SyDirection is the vertical direction of focal line.Pass through the y in sample
Direction is scanned, and synchronizes extension detection (different from conventional detection scheme, to be herein two-dimensional detection), can get sample
The generated optical signal in excitation line.
Calculating reconstruction is carried out to the two-dimensional signal, the high-quality signal in sample in excitation line can be recovered.Change vibration
The deflection angle of mirror can get the optical signal at longitudinal (y-axis) different location.In addition, using the technology axial chromatography ability into
Row axial scan (relative position for changing mobile example and microcobjective), can get the optical signal of sample different depth；Into
One step, carrying out delayed data acquisition using the high speed micro-imaging ability of the technology, (i.e. the dynamic of acquisition different moments is believed
Breath), it can get (x, y, z, t) four-dimensional information of sample.
A kind of light microscopic imaging device focused based on line scanning space-time that the embodiment of the present invention is proposed can effectively be pressed down
Signal cross-talk caused by stray light processed has many advantages, such as wide visual field, high spatial resolution, high time resolution, deep penetration depth,
Abundant information can be provided for biological dynamic process research, medical diagnosis on disease foundation etc..
Fig. 3 is a kind of structural representation that the light microscopic imaging device that space-time focuses is scanned based on line of one embodiment of the invention
Figure, as shown in figure 3, the device include laser emitting module 301, optical beam transformation module, line scan module, optical microphotograph module,
Synchronous filtering module rebuilds module, and sample is placed on sample stage 315.
Wherein, laser emitting module 301 uses femto-second laser (such as Coherent Chameleon Discovery system
Column), optical beam transformation module uses the Kepler telescope system being made of lens 302 and cylindrical lens 303 (for 4f system)；Line is swept
Retouching module includes transmission grating 304, lens 305 and scanning galvanometer 307；Optical microphotograph module includes two lens 308,309 and
Microcobjective 310；Synchronous filtering module includes dichroic mirror 306, low pass filters 311, lens 312 and two-dimensional area detector 313
(using sCMOS or EMCCD).
The relative positional relationship of above-mentioned component are as follows:
Lens 302 constitute 4f system with cylindrical lens 303 and are expanded, and transmission grating 304 is placed in the image planes of cylindrical lens 303
Transmission grating 304 is imaged at scanning galvanometer 307 by place, lens 305, and lens 308 and 309 constitute 4f system and make scanning galvanometer
307 are conjugated with the entrance pupil face of microcobjective 310, and lens 312 and lens 308 constitute 4f system and object plane made to be imaged on two-dimensional surface spy
Survey device 313.
Computer 314 is also illustrated in Fig. 3, is detected for controlling the deflection angle of scanning galvanometer 307, and to two-dimensional surface
The collected two-dimensional signal of device 313 carries out calculating reconstruction.
The laser beam that ultra-short pulse laser light source 301 is issued in the embodiment expands and (changes through lens 302, cylindrical lens 303
Become laser beam diameter) after be incident on transmission grating 304, under the action of transmission grating 304 ultrashort pulsed beam generate angle
Dispersion (purpose is so that the light beam of the introducing angle dispersion is full of the back focal plane of object lens after subsequent optical element), through lens 305
It is projected on scanning galvanometer 307 after collimation through dichroic mirror 306 and introduces variable deflection angle (deflection angle is by galvanometer driving voltage
Driving sets deflection angle according to scanning area), in the biological sample via lens 308,309 and microcobjective 310 finally
Focal line is generated on object lens focal plane.
The reverse transfer after the collection of microcobjective 310 of the optical signal as caused by nonlinear optical effect, successively by saturating
Mirror 309,308 and scanning galvanometer 307, and reflected by dichroic mirror 306.Later, which successively passes through low pass filters
311, lens 312 finally enter two-dimensional area detector 313 and carry out 2D signal detection, and it is extensive to carry out calculating reconstruction by computer 314
It appears again one-dimension information.
It should be noted that the frame trigger signal of row the scanning trigger signal and two-dimensional area detector 313 of scanning galvanometer 307
It is synchronous.
By adopting the above technical scheme, it can get biological sample along the information of focal line.Driving scanning galvanometer 307 carries out longitudinal direction
(y-axis) scanning, can get two-dimensional signal of the biological sample in focusing surface.
Axial scan is carried out by mobile microcobjective 310, can get the information at sample three-dimensional different depth.If carrying out
Delayed data acquisition, can get (x, y, z, t) four-dimensional information of sample.
Fig. 4 is a kind of structural representation that the light microscopic imaging device that space-time focuses is scanned based on line of further embodiment of this invention
Figure, as shown in figure 4, being to be additionally arranged quality of the adaptive optical element to improve focal line with the difference of Fig. 3 in Fig. 4.
The present embodiment device includes laser emitting module and optical beam transformation module, line scan module, optical microphotograph module, same
It walks filter module, rebuild module, biological sample is placed on sample stage 420；Wherein, laser emitting module 401 is swashed using femtosecond
Light device (such as Coherent Chameleon Discovery series), optical beam transformation module is used by lens 402 and cylindrical lens 403
The Kepler telescope system of composition (for 4f system)；
The line scan module focused based on space-time includes transmission grating 404, five lens 405,407,408,410,411,
The adaptive optical element 406 (the present embodiment uses spatial light modulator) being set between lens 405 and 407, scanning galvanometer
412；Optical microphotograph module includes two lens 413,414, microcobjective 415；Synchronous filtering module includes dichroic mirror 409, low
Pass filter piece 416, ordinary lens 417, two-dimensional area detector 418 (use sCMOS or EMCCD).
The relative positional relationship of above-mentioned component are as follows:
Lens 402 constitute 4f system with cylindrical lens 403 and are expanded, and transmission grating 404 is placed in the image planes of cylindrical lens 403
Transmission grating 404 is imaged at spatial light modulator 406 by place, lens 405, lens 407 and 408, lens 410 and 411, lens
413 and 414, which respectively constitute 3 groups of concatenated 4f systems, makes spatial light modulator 406 and scanning galvanometer 412 and microcobjective 415
Entrance pupil face conjugation, lens 417 and 410 composition 4f system make object plane be imaged on two-dimensional area detector 418.
Computer 419 is also illustrated in Fig. 4, is detected for controlling the deflection angle of scanning galvanometer 412, and to two-dimensional surface
The collected two-dimensional signal of device 418 carries out calculating reconstruction.
In the embodiment, the laser beam that laser emitting module 401 is issued is incident after lens 402, cylindrical lens 403 expand
To transmission grating 404, the ultrashort pulsed beam generates angle dispersion under the action of transmission grating 404, throws after the collimation of lens 405
It is mapped to spatial light modulator 406 and carries out spectrum phase shaping, further to overcome the influence of tissue scatter, later successively by saturating
Mirror 407, lens 408, dichroic mirror 409, lens 410, lens 411 introduce variable deflection angle on scanning galvanometer 412, finally via
Focal line is generated on the object lens focal plane of lens 413,414 and microcobjective 415 in the biological sample.
The reverse transfer after the collection of microcobjective 415 of the optical signal as caused by nonlinear optical effect, successively by saturating
Mirror 414, lens 413, scanning galvanometer 412, lens 411, lens 410, and reflected by dichroic mirror 409.
Later, which successively passes through low pass filters 416, lens 417, finally enters two-dimensional area detector 418
2D signal detection is carried out, calculating reconstruction is carried out by computer 419 and recovers one-dimension information.
It should be noted that the frame trigger signal of row the scanning trigger signal and two-dimensional area detector 419 of scanning galvanometer 412
It is synchronous；Spatial light modulator 406 is using the measurement wavefront distortion of adaptive optics method and to apply compensation wavefront, it is ensured that focal line
High axial resolution.By adopting the above technical scheme, it can get biological sample along the information of focal line.
Driving scanning galvanometer 412 carries out longitudinal direction (y-axis) scanning, can get two-dimensional signal of the biological sample in focusing surface.
Axial scan is carried out by mobile microcobjective 415, can get the information at sample three-dimensional different depth.
If carrying out delayed data acquisition, (x, y, z, t) four-dimensional information of sample can get.
In actual experiment, it is contemplated that required excitation light power is likely larger than the light injury threshold of spatial light modulator, may be used also
It can need to increase lens group before spatial light modulator 406, so that light beam is expanded on the direction extended perpendicular to spectrum
Light intensity is lower than the light injury threshold of spatial light modulator and following.
Fig. 5 is a kind of flow chart that the light micro imaging method that space-time focuses is scanned based on line of the embodiment of the present invention, such as Fig. 5
It is shown, this method comprises:
S1 generates nonlinear excitation pulse light；
S2, obtains the angle dispersion and deflection angle of the excitation pulse light, and the excitation pulse light carries out sample
Scanning；
S3 forms the focal line focused simultaneously in two dimensions in space and time in the sample, based on non-linear
Optical effect excites the sample to generate transmitting optical signal；
S4 detects the transmitting optical signal of the sample, and is filtered to the transmitting optical signal, the synchronous filtering mould
The trigger signal of block is synchronous with the trigger signal of the line scan module；
S5 carries out 2-d reconstruction according to the transmitting optical signal that the synchronous filtering module detects, obtains the sample
Reconstructed image.
A kind of raising line that the embodiment of the present invention proposes sweeps the method that space-time focuses micro-imaging depth, specifically includes following
Step:
1) parameter setting: along sample, (setting laser focuses institute for lateral, longitudinal direction and axial respectively x-axis, y-axis and z-axis for setting
The excitation line direction of formation is transverse direction, i.e. x-axis；Scanning direction is longitudinal direction, i.e. y-axis), it sets along time dimension direction as t axis；If
The galvanometer deflection angle step-length scanned along sample vertical line is realized in target scan areas XYZ in random sample product, setting, and edge is realized in setting
The microcobjective axial direction step-length of sample axial scan sets imaging collection period according to the size of target scan areas and scanning is total
Duration；
2) ultra-short pulse laser is generated using ultra-short pulse laser light source；
3) it in start time scan period, is formed in the sample by space-time focus method simultaneously in space and time
The excitation line (in the x-direction, can realize that the direction y of focal line is scanned by changing the deflection angle of galvanometer) that two dimensions focus；
4) optical signals are excited on the focal line of step 3) by nonlinear optical effect, the optical signal is via micro- object
Reverse transfer after mirror is collected, again via the ultra-short pulse laser for filtering out reflection after galvanometer by filter plate；
5) it is extended detection by being placed in the planar array detector in sample at the conjugate position of focal line, i.e., to excitation line
Optical signal caused by upper carries out (Sx,Sy) two-dimensional detection；
6) (the S that will be detectedx,Sy) two-dimensional signal carries out calculating reconstruction；
7) deflection angle scanned according to the galvanometer deflection angle step-size change line of setting obtains different longitudinal (y-axis) positions of sample
The optical signal at place is set, until the scanning traversal target area XY；Wherein, planar array detector is synchronous with the trigger signal of galvanometer；
8) it according to the depth of the microcobjective axial direction step-size change focal line of setting, obtains at different depth (z-axis) position
Optical signal, until scanning traversal the target area XYZ；Present scanning cycle terminates, and executes step 9)；
9) step 3)-step 6) is repeated according to the imaging acquisition period of setting and carries out delay micro-imaging, set until reaching
Fixed scanning total duration completes line scanning space-time and focuses micro-imaging.
A kind of light micro imaging method focused based on line scanning space-time provided in an embodiment of the present invention, can effectively inhibit
Signal cross-talk caused by stray light has many advantages, such as wide visual field, high spatial resolution, high time resolution, deep penetration depth, can
Abundant information is provided for biological dynamic process research, medical diagnosis on disease foundation etc..
The apparatus embodiments described above are merely exemplary, wherein described, unit can as illustrated by the separation member
It is physically separated with being or may not be, component shown as a unit may or may not be physics list
Member, it can it is in one place, or may be distributed over multiple network units.It can be selected according to the actual needs
In some or all of the modules achieve the purpose of the solution of this embodiment.Those of ordinary skill in the art are not paying creativeness
Labour in the case where, it can understand and implement.
Through the above description of the embodiments, those skilled in the art can be understood that each embodiment can
It realizes by means of software and necessary general hardware platform, naturally it is also possible to pass through hardware.Based on this understanding, on
Stating technical solution, substantially the part that contributes to existing technology can be embodied in the form of software products in other words, should
Computer software product may be stored in a computer readable storage medium, such as ROM/RAM, magnetic disk, CD, including several fingers
It enables and using so that a computer equipment (can be personal computer, server or the network equipment etc.) executes each implementation
Method described in certain parts of example or embodiment.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (9)
1. a kind of light microscopic imaging device focused based on line scanning space-time characterized by comprising
Laser emitting module, for generating nonlinear excitation pulse light；
Line scan module for obtaining the angle dispersion and deflection angle of the excitation pulse light, and makes the excitation pulse light pair
Sample is scanned；
Optical microphotograph module, for forming the focal line focused simultaneously in two dimensions in space and time in the sample, with
The sample is excited to generate transmitting optical signal based on nonlinear optical effect；
Synchronous filtering module is filtered for detecting the transmitting optical signal of the sample, and to the transmitting optical signal, described
The trigger signal of synchronous filtering module is synchronous with the trigger signal of the line scan module；
Rebuild module, transmitting optical signal for detecting according to the synchronous filtering module carries out 2-d reconstruction, described in acquisition
The reconstructed image of sample.
2. device according to claim 1, which is characterized in that further include optical beam transformation module, optical beam transformation module position
Before the line scan module, the optical beam transformation module is used to adjust the beam sizes of the excitation pulse light.
3. device according to claim 1, which is characterized in that the line scan module include diffractive-optical element, lens and
Optical scanning element, the diffractive-optical element are located at the object space focal plane of the lens, and the optical scanning element is located at described
The image space focal plane of lens, the diffractive-optical element are used to obtain the angle dispersion of excitation pulse light adjusted, and the optics is swept
Element is retouched for obtaining the variable deflection angle of excitation pulse light adjusted.
4. device according to claim 3, which is characterized in that the line scan module further includes adaptive optical element, institute
It states adaptive optical element to be located at before the lens, for carrying out shaping to the spectrum phase of excitation pulse adjusted.
5. device according to claim 3, which is characterized in that the optical microphotograph module includes lens group and microcobjective,
The lens group constitutes 4f system, and the entrance pupil face of the optical scanning element and the optical microscopy is conjugated.
6. device according to claim 5, which is characterized in that further include: dispersion pre-compensation module, the dispersion pre-compensation mould
Block reaches the dispersion accumulated before the microcobjective focusing surface for compensating the excitation pulse light.
7. device according to claim 1, which is characterized in that the laser emitting module be femtosecond pulse laser or picosecond
Pulse laser.
8. device according to claim 1, which is characterized in that the optical beam transformation module is Galilean telescope or Kepler
Telescope.
9. a kind of light micro imaging method focused based on line scanning space-time characterized by comprising
Generate nonlinear excitation pulse light；
The angle dispersion and deflection angle of the excitation pulse light are obtained, and the excitation pulse light is scanned sample；
The focal line focused simultaneously in two dimensions in space and time is formed, in the sample to be based on nonlinear optical effect
The sample is excited to generate transmitting optical signal；
The transmitting optical signal of the sample is detected, and the transmitting optical signal is filtered, the touching of the synchronous filtering module
It signals synchronous with the trigger signal of the line scan module；
2-d reconstruction is carried out according to the transmitting optical signal that the synchronous filtering module detects, obtains the reconstruct image of the sample
Picture.
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| CN112304897A (en) * | 2020-09-14 | 2021-02-02 | 中国人民解放军战略支援部队航天工程大学 | Spectrum selection method and system for combustion field two-dimensional reconstruction |
| WO2022261969A1 (en) * | 2021-06-18 | 2022-12-22 | 合圣科技股份有限公司 | Image capture system and image capture method |
| WO2023064374A1 (en) * | 2021-10-13 | 2023-04-20 | Fluorescence Microscopy Business Unit (FMBU) of Bruker Nano Surfaces Division | Method and apparatus for rapid depth-scanning in laser imaging |
| CN116105644A (en) * | 2023-04-10 | 2023-05-12 | 清华大学 | Radiation scanning imaging method and radiation processing method |
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