CN108742532A - The wide visual field chromatography ultraphotic spectrum micro imaging method and device focused based on space-time - Google Patents
The wide visual field chromatography ultraphotic spectrum micro imaging method and device focused based on space-time Download PDFInfo
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Abstract
The present invention discloses a kind of wide visual field chromatography ultraphotic spectrum micro imaging method and device focused based on space-time, belongs to microspectrum imaging and technical field of analytical chemistry.This method generates ultra-short pulse laser using ultra-short pulse laser light source, it focused by using space-time and generates focal line in the sample, collect excited fluorescence and stray light is filtered out using confocal optics slit, acquisition fluorescence spectrum information completes the spectral information (x of sample, λ) obtain, finally sample (x is obtained with delayed sweep by three dimensions scanning, λ, y, z, t) five-dimensional information.Linear scanning system, optical microscope system and the filtering and synchronous spectrum confocal detection system that the present apparatus includes ultra-short pulse laser light source and beam shaping system, is focused based on space-time, and filter obtained with the spectral information in synchronous spectrum confocal detection system it is synchronous with the combination linear scanning system centerline scan trigger signal of space-time focusing technology.The present invention has many advantages, such as wide visual field, high spatial resolution, high time resolution, high spectral resolution.
Description
Technical field
The present invention relates to a kind of wide visual field chromatography ultraphotic spectrum micro imaging methods and device focused based on space-time, belong to aobvious
Micro- light spectrum image-forming and technical field of analytical chemistry.
Background technology
Ultraphotic spectrum micro-imaging (Hyperspectral Microscopy) has important answer in field of biomedical research
With the fields such as image-guidance are increasingly subject to the extensive attention of people especially in clinical disease diagnosis, art.It is aobvious using ultraphotic spectrum
Micro- imaging technique obtains the distinguishable spectral information in space, can be provided for medical diagnosis on disease the physiological parameter of biological tissue, pattern and
The information such as component.Currently, composing micro-imaging technique using ultraphotic, the Noninvasive testing to kinds cancer can have been realized.
In essence, ultraphotic spectrum micro-imaging technique is that more high dimensional information (i.e. light is obtained on the basis of micro-imaging
Spectrum information) technology.According to the realization method of micro-imaging technique, current ultraphotic spectrum micro-imaging technique can be divided into based on general
The micro- ultraphotic spectrum micro-imaging technique in logical wide field, the ultraphotic spectrum micro-imaging technique etc. based on confocal scanning.The former can be fast
The fast parallel spectral information obtained in wide visual field, but it is limited to that common wide field is micro- not to be had chromatography ability, easily made by tissue scatter
The shortcomings of at signal cross-talk, the technology are only applicable to transparent organism sample.The latter is based on focusing principle altogether, inhibits to a certain extent
The influence of tissue scatter and obtain axial resolution, but due to needing to carry out point by point scanning imaging, image taking speed by
Limitation.Micro-imaging technique is composed based on the micro- ultraphotic of mating plate in addition, having also appeared in recent years, regrettably the technology is not equally
Suitable for scattering imaging of tissue.
In order to overcome the influence of biological tissue scatters and improve imaging penetration depth, people are by nonlinear optics microtechnic
It is introduced into ultraphotic spectrum micro-imaging, has developed the ultraphotic spectrum microtechnic based on nonlinear optical effect, and be widely used in
Biomedical research.Since common nonlinear optics microtechnic still uses spot scan mode to overcome the shadow of tissue scatter mostly
It rings, the speed and current potential of imaging must be affected.On the other hand, it is eliminated although the nonlinear optics excited using face is micro-
Speed bottle-neck caused by point by point scanning, but institute's excitation signal crosstalk after tissue scatter is serious, be unsuitable for scattering tissue spectrum at
Picture.It can be influenced therebetween in image taking speed with scattering is inhibited using the common nonlinear optics microtechnic of line scan mode
Compromise, but the axial resolution that this method is obtained relative to spot scan mode reduces, also nonideal selection.
Therefore, at present a technical problem that is urgently needed by the technical personnel in the field at present is that：How innovatively
A kind of effective measures are proposed, to solve the deficiencies in the prior art.
Invention content
The purpose of the invention is to overcome the shortcoming of prior art, a kind of wide visual field focused based on space-time is provided
Chromatograph ultraphotic spectrum micro imaging method and device.The present invention be suitable for scattering imaging in biological tissues, can be improved image taking speed and
Flux, and there is high axial resolution.
To achieve the goals above, the present invention adopts the following technical scheme that：
A kind of wide visual field chromatography ultraphotic focused based on space-time proposed by the present invention composes micro imaging method, and feature exists
In including the following steps：
1) parameter setting：Setting lateral, longitudinal direction and axial respectively x-axis, y-axis and z-axis along sample, set along laser spectrum
Direction is λ axis, is set along time dimension direction as t axis；The target scan areas XYZ in sample is set, setting is realized along sample
The galvanometer deflection angle step-length of vertical line scanning, the microcobjective axial direction step-length along sample axial scan is realized in setting, according to target
The size setting spectral information collection period and scanning total duration of scanning area；
2) ultra-short pulse laser light source is utilized to generate ultra-short pulse laser；
3) it in start time scan period, is formed in the sample by space-time focus method simultaneously in space and time
The focal line that two dimensions focus；
4) fluorescence signal is inspired on the focal line of step 3) by nonlinear optical effect, the fluorescence signal is via aobvious
Reverse transfer after speck mirror is collected, then filters out the ultra-short pulse laser of reflection by filter plate and filters out sample by confocal optics slit
Stray light caused by product scattering obtains the bar shaped transmitting fluorescence of sample；
5) obtained bar shaped is emitted by fluorescence by dispersion element and carries out spectral evolution, spectrum letter is carried out by planar array detector
Breath acquisition, obtains (x, λ) two-dimensional signal of sample；Meanwhile the deflection scanned according to the galvanometer deflection angle step-size change line of setting
Angle obtains (x, λ, y) three-dimensional information of sample, until the target areas scanning traversal XY, complete sample two dimensional surface different location
The acquisition of spectral information；Wherein, the exhibition of the bar shaped transmitting fluorescence is completely covered in the search coverage size of the planar array detector
Degree is opened, and the planar array detector is synchronous with the trigger signal of galvanometer；
6) according to the depth of the microcobjective axial direction step-size change focal line of setting, (x, λ, the y, z) for obtaining sample is four-dimensional
Information obtains in sample three dimensions not until scanning traversal XYZ target areas, complete the acquisition of different depth spectral information
With the spectral information of position；Present scanning cycle terminates, and executes step 7)；
7) step 3)~step 6) is repeated according to the spectral information collection period of setting and composes micro-imaging into line delay ultraphotic,
(x, λ, y, z, t) five-dimensional information of sample is obtained, until reaching the scanning total duration of setting, it is aobvious to complete wide visual field chromatography ultraphotic spectrum
Micro- imaging.
The present invention also proposes that a kind of wide visual field chromatography ultraphotic focused based on space-time according to above-mentioned composes micro imaging method
Device, which is characterized in that the linear scanning system that is focused including ultra-short pulse laser light source and beam shaping system, based on space-time,
Optical microscope system and filtering and synchronous spectrum confocal detection system；Wherein,
The ultra-short pulse laser light source and beam shaping system, ultra-short pulse laser light source are non-linear for providing generation
The excitation pulse light of optical signalling, beam shaping system is for adjusting the excitation pulse light beam sizes；
The linear scanning system focused based on space-time, is placed in after the beam shaping system, including optical diffraction member
Part, lens and optical scanning element, the diffractive-optical element and the optical scanning element are respectively placed in the object of the lens
Square focal plane and image space focal plane；The diffractive-optical element is used to introduce the angle dispersion of excitation pulse light beam, the optical scanner member
Part is used to introduce the variable deflection angle of excitation pulse light beam；
The optical microscope system is placed in after the optical scanning element, including lens group and microcobjective, by described
Lens group connects entrance pupil face after the optical scanning element and microcobjective, constitutes 4f systems, which is used for
The focal line focused simultaneously in two dimensions in space and time is formed in sample, to excite tissue sample and be based on nonlinear optics
Effect generates transmitting fluorescence；
The filtering and synchronous spectrum confocal detection system, are placed in the linear scanning system focused based on space-time and are emitted
Fluorescence through the microcobjective collect and reverse transfer optical scanning element after, including filter plate, confocal optics slit and
Spectrometer, transmitting fluorescence signal for selecting sample simultaneously carry out spectral information acquisition；The acquisition of information of the spectrometer and institute
The trigger signal for stating the linear scanning system focused based on space-time is synchronized.
Compared with prior art, the present invention has the following advantages：Skill is scanned by using the line based on space-time focus method
Art, and propose corresponding synchronous spectrum confocal detection technology, it is ensured that high axial resolution, low scattered signal crosstalk and high-speed light
Spectrum information obtains, and the wide visual field chromatography ultraphotic spectrum micro-imaging technique realized chromatographs aobvious suitable for deep tissues wide visual field high speed
Micro- light spectrum image-forming.
Generally, proposed wide visual field chromatography ultraphotic spectrum micro-imaging technique can be used for deep layer biological tissue (x,
Y, z, t, λ) five-dimensional information acquisition, have many advantages, such as wide visual field, high spatial resolution, high time resolution, high spectral resolution.
Description of the drawings
Fig. 1 is the structural schematic diagram of wide visual field chromatography ultraphotic spectrum microscopic imaging device of the present invention.
Fig. 2 is the principle of the present invention schematic diagram.
Fig. 3 is the structural schematic diagram of apparatus of the present invention embodiment 1.
Fig. 4 is the structural schematic diagram of apparatus of the present invention embodiment 2.
Specific implementation mode
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, below in conjunction with the accompanying drawings and specific real
Applying mode, the present invention will be further described in detail.
A kind of wide visual field chromatography ultraphotic proposed by the present invention composes micro imaging method, specifically includes following steps：
1) parameter setting：Setting lateral, longitudinal direction and axial respectively x-axis, y-axis and z-axis along sample, set along laser spectrum
(i.e. wavelength) direction is λ axis, is set along time dimension direction as t axis；The target scan areas XYZ in sample is set, setting is real
Now along the galvanometer deflection angle step-length of sample vertical line scanning, the microcobjective axial direction step-length along sample axial scan is realized in setting,
Spectral information collection period and scanning total duration are set according to the size of target scan areas；
2) ultra-short pulse laser light source is utilized to generate ultra-short pulse laser；
3) it in start time scan period, is formed in the sample by space-time focus method simultaneously in space and time
The focal line (in the x-direction, can realize that the directions y of focal line are scanned by changing the deflection angle of galvanometer) that two dimensions focus；
4) fluorescence signal is inspired on the focal line of step 3) by nonlinear optical effect, the fluorescence signal is via aobvious
Reverse transfer after speck mirror is collected, then filters out the ultra-short pulse laser of reflection by filter plate and filters out sample by confocal optics slit
Stray light caused by product scattering obtains the bar shaped transmitting fluorescence of sample；
5) obtained bar shaped is emitted by fluorescence by dispersion element and carries out spectral evolution, spectrum letter is carried out by planar array detector
Breath acquisition, obtains (x, λ) two-dimensional signal of sample；Meanwhile the deflection scanned according to the galvanometer deflection angle step-size change line of setting
Angle obtains (x, λ, y) three-dimensional information of sample, until the target areas scanning traversal XY, complete sample two dimensional surface different location
The acquisition of spectral information；Wherein, the expansion degree of bar shaped transmitting fluorescence is completely covered in the search coverage size of planar array detector, and
Planar array detector is synchronous with the trigger signal of galvanometer；
6) according to the depth of the microcobjective axial direction step-size change focal line of setting, (x, λ, the y, z) for obtaining sample is four-dimensional
Information obtains in sample three dimensions not until scanning traversal XYZ target areas, complete the acquisition of different depth spectral information
With the spectral information of position；Present scanning cycle terminates, and executes step 7)；
7) step 3)~step 6) is repeated according to the spectral information collection period of setting and composes micro-imaging into line delay ultraphotic,
(x, λ, y, z, t) five-dimensional information of sample is obtained, until reaching the scanning total duration of setting, it is aobvious to complete wide visual field chromatography ultraphotic spectrum
Micro- imaging.
The present invention proposes that a kind of wide visual field chromatography ultraphotic composes microscopic imaging device, structure such as Fig. 1 also according to the above method
It is shown, including：Ultra-short pulse laser light source and beam shaping system, the linear scanning system based on space-time focusing, optical microphotograph system
System and filtering and synchronous spectrum confocal detection system；Wherein,
Ultra-short pulse laser light source and beam shaping system, ultra-short pulse laser light source generate nonlinear optics for providing
The excitation pulse light of signal, beam shaping system is for adjusting excitation pulse light beam sizes；
Based on space-time focus linear scanning system, be placed in after above-mentioned beam shaping system, including diffractive-optical element, thoroughly
Mirror and optical 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；
Diffractive-optical element is used to introduce the angle dispersion of excitation pulse light beam, and what optical scanning element was used to introduce excitation pulse light beam can
Become deflection angle；
Optical microscope system 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 systems, 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 fluorescence based on nonlinear optical effect；
Filtering and synchronous spectrum confocal detection system are placed in the linear scanning system focused based on space-time and emit fluorescence through aobvious
Speck mirror is collected and after the optical scanning element of reverse transfer, including filter plate, confocal optics slit and spectrometer, for selecting
Go out the transmitting fluorescence signal of sample and carries out spectral information acquisition；Wherein, the acquisition of information of spectrometer with based on space-time focus
The trigger signal of linear scanning system synchronizes.
Further, it in ultra-short pulse laser light source and beam shaping system, is also set before ultra-short pulse laser output
There is 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
Object sample scattering influence caused by being ultimately imaged.
The specific implementation of each component part is as follows in apparatus of the present invention：
In ultra-short pulse laser light source and beam shaping system, 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,
The ultra-short pulse laser light source of fixed wave length or the ultra-short pulse laser light source of tunable wavelength can be selected；Beam shaping system is
Galilean telescope system or Kepler telescope system.It is non-thread that ultra-short pulse laser light source and beam shaping system provide generation
In the exciting light of property optical signalling non linear optical signal by two-photon absorption fluorescent effect, Three-photon absorption fluorescent effect or
Any one of two-photon excitation-fluorescence resonance energy transfer effect generates.
In linear scanning system based on space-time focusing technology, grating, distorting lens, spatial light tune can be selected in diffractive-optical element
Device processed or other diffractive-optical elements；Optical scanning element selects galvanometer, polygonal mirror or acousto-optic modulator etc..
Filtering with synchronous spectrum confocal detection system in, filter plate select dichroic mirror, band pass filter, low pass filters or
Long pass filter piece.Confocal optics slit is placed in the conjugate planes in biological sample excitation face, and the width of the confocal optics slit is by sample
The design size of conjugate image determines.Spectrometer is made of dispersion element, two-dimensional area detector and two lens, and the first lens will be total to
Burnt optical slot constitutes object-image relation with dispersion element, and dispersion element and two-dimensional area detector are constituted image and closed by the second lens
System；Prism, grating or other dispersion elements can be selected in dispersion element；Two-dimensional area detector selects charge coupled cell (CCD), electricity
Sub- multiplying charge coupling element (EMCCD) or Scientific Grade complementary mos device (sCMOS) etc..
With reference to Fig. 2, the principle of the present invention schematic diagram is shown.Using nonlinear optical effect and combine space-time focusing technology
(U.S. patent Nos US20080151238A1) can generate the focal line (side x with high axial resolution on biological sample
To), the fluorescence spectrum signal excited is imaged through confocal detection on the 2 D photoelectric test surface (Dx, Dy) of spectrometer, wherein
The directions Dy are spectrum lambda dimension.It is scanned by the directions y in sample, and synchronizes confocal detection, can get sample and exist
(x, λ, y) three-dimensional information under wide visual field.Axial scan, which is carried out, using the axial chromatography ability of the technology (changes mobile sample
With the relative position of microcobjective), it can get (x, λ, y, z) four-dimensional information of sample；Further, the high speed of the technology is utilized
Spectrum micro-imaging ability carries out delayed data acquisition (sample (x, λ, y, the z) four-dimensional information for acquiring different moments), can obtain
Obtain (x, λ, y, z, t) five-dimensional information of sample.As it can be seen that wide visual field chromatography ultraphotic spectrum micro imaging method proposed by the invention and
Its device has many advantages, such as wide visual field, high spatial resolution, high time resolution, high spectral resolution, can be biodynamic mistake
Journey research, medical diagnosis on disease foundation etc. provide abundant information.
Embodiment 1：
Referring to Fig. 3, the wide visual field chromatography ultraphotic for describing the present embodiment in detail composes microscopic imaging device, which includes
Ultra-short pulse laser light source and beam shaping system, the linear scanning system focused based on space-time, optical microscope system, filtering with it is same
Spectral Confocal detection system is walked, biological sample is placed on sample stage 319.Wherein, ultra-short pulse laser light source and optical beam transformation
Ultra-short pulse laser light source 301 in system is using femto-second laser (such as Coherent Chameleon Discovery systems
Row), beam shaping system uses the Kepler telescope system being made of lens 302 and cylindrical lens 303 (for 4f systems)；It is based on
The linear scanning system that space-time focuses includes transmission grating 304, lens 305 and scanning galvanometer 307；Optical microscope system includes two
Lens 308,309 and microcobjective 310；Filtering and synchronous spectrum confocal detection system include dichroic mirror 306, low pass filters
311, lens 312, confocal optics slit 313 (optical slot width is determined by the design size of biological sample conjugate image), and
By two lens 314,316,317 spectrometer constituted of reflecting grating 315 and two-dimensional area detector (using sCMOS or EMCCD).
The relative position relation of above-mentioned component is：Lens 302 constitute 4f systems with cylindrical lens 303 and are expanded, and transmission grating 304 is set
At the image planes of cylindrical lens 303, transmission grating 304 is imaged at scanning galvanometer 307 by lens 305, and lens 308 and 309 are constituted
4f systems make scanning galvanometer 307 and the entrance pupil face of microcobjective 310 be conjugated, and lens 312 constitute 4f systems with lens 308 and make
Object plane is imaged at confocal optics slit 313, and low pass filters 311 are tightly placed in before lens 312, and lens 314 are by confocal optics
Slit 313 is imaged on reflecting grating 315, and reflecting grating 315 is imaged on two-dimensional area detector 317 by lens 316.Also show in Fig. 3
It has anticipated out computer 318, the deflection angle for controlling scanning galvanometer 307, and to 317 collected spectrum of two-dimensional area detector
Information carries out conventional image reconstruction and data processing.
The laser beam that ultra-short pulse laser light source 301 is sent out 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 projects on scanning galvanometer 307 and introduces through dichroic mirror 306 after collimation and can be changed 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 optical signal caused by nonlinear optical effect reversely passes after the collection of microcobjective 310
It is defeated, pass through lens 309,308 and scanning galvanometer 307 successively, and reflected by dichroic mirror 306.Later, which passes through successively
Low pass filters 311, lens 312 and confocal optics slit 313 finally enter spectrometer and carry out signal collection.It should be noted that
It is that the row scanning trigger signal of scanning galvanometer 307 is synchronous with the frame trigger signal of two-dimensional area detector 317.Using above-mentioned technology
Scheme can get (x, λ, y) three-dimensional information of biological sample.Axial scan is carried out by mobile microcobjective 310, can get sample
This (x, λ, y, z) four-dimensional information.If carrying out delayed data acquisition, (x, λ, y, z, t) five-dimensional information of sample can get.
Embodiment 2：
Referring to Fig. 4, the wide visual field chromatography ultraphotic for describing the present embodiment in detail composes microscopic imaging device, the present embodiment with
Difference lies in be additionally arranged adaptive optical element for embodiment 1.The present embodiment device includes ultra-short pulse laser light source and light beam
Transformation system, linear scanning system, optical microscope system, filtering and the synchronous spectrum confocal detection system focused based on space-time, it is raw
Object sample is placed on sample stage 424；Wherein, the ultra-short pulse laser light in ultra-short pulse laser light source and beam shaping system
Using femto-second laser (such as Coherent Chameleon Discovery series), beam shaping system is used by saturating in source 401
The Kepler telescope system that mirror 402 and cylindrical lens 403 are constituted (for 4f systems)；Based on space-time focus linear scanning system include
Transmission grating 404, five lens 405,407,408,410,411, the adaptive optics member being set between lens 405 and 407
Part 406 (the present embodiment uses spatial light modulator), scanning galvanometer 412；Optical microscope system includes two lens 413,414,
Microcobjective 415；Filtering and synchronous spectrum confocal detection system include dichroic mirror 409, low pass filters 416, ordinary lens
417, confocal optics slit 418 (optical slot width is determined by the design size of biological sample conjugate image), and thoroughly by two
422 spectrometer constituted of mirror 419,421, reflecting grating 420 and two-dimensional area detector (using sCMOS or EMCCD).Above-mentioned member device
The relative position relation of part is：Lens 402 constitute 4f systems with cylindrical lens 403 and are expanded, and transmission grating 404 is placed in cylindrical lens
At 403 image planes, transmission grating 404 is imaged at spatial light modulator 406 by lens 405, lens 407 and 408, lens 410
3 groups of concatenated 4f systems, which are respectively constituted, with 411, lens 413 and 414 makes spatial light modulator 406 and scanning galvanometer 412 and aobvious
The entrance pupil face of speck mirror 415 is conjugated, and lens 417 and 410 constitute 4f systems and object plane is imaged at confocal optics slit 418,
Low pass filters 416 are tightly placed in before lens 417, and confocal optics slit 418 is imaged on reflecting grating 420, lens by lens 419
Reflecting grating 420 is imaged on two-dimensional area detector 422 by 421.Computer 423 is also illustrated in Fig. 4, is shaken for controlling scanning
The deflection angle of mirror 412, and 422 collected spectral information of two-dimensional area detector is carried out at conventional image reconstruction and data
Reason.
In the embodiment, the laser beam that ultra-short pulse laser light source 401 is sent out is after lens 402, cylindrical lens 403 expand
It is incident on transmission grating 404, the ultrashort pulsed beam generates angle dispersion under the action of transmission grating 404, is collimated through lens 405
After project spatial light modulator 406 carry out spectrum phase shaping passed through successively later with further overcoming the influence of tissue scatter
It crosses lens 407, lens 408, dichroic mirror 409, lens 410, lens 411 and introduces variable deflection angle on scanning galvanometer 412, finally
Via generating focal line on the object lens focal plane of lens 413,414 and microcobjective 415 in the biological sample.It is imitated by nonlinear optics
The reverse transfer after the collection of microcobjective 415 of fluorescence signal caused by answering passes through lens 414, lens 413, scans and shake successively
Mirror 412, lens 411, lens 410, and reflected by dichroic mirror 409.Later, the signal beams successively pass through low pass filters 416,
Lens 417 and confocal optics slit 418 finally enter spectrometer and carry out signal collection.It should be noted that scanning galvanometer 412
Row scanning trigger signal it is synchronous with the frame trigger signal of two-dimensional area detector 422；Spatial light modulator 406 is using adaptive
Optical method for measuring wavefront distortion simultaneously applies compensation wavefront (being carried out before light spectrum image-forming).Using above-mentioned technical proposal, can get
(x, λ, y) three-dimensional information of biological sample.Carry out axial scan by mobile microcobjective 415, can get biological sample (x,
λ, y, z) four-dimensional information.If carrying out delayed data acquisition (i.e. multi collect), (x, λ, y, the z, t) five that can get biological sample is tieed up
Information.
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 that spectrum extends
Light intensity is less than the light injury threshold of spatial light modulator and following.
To sum up, the present invention is by combining space-time focusing technology into line scanning technique, and proposes corresponding synchronous spectrum
Confocal detection technology ensure that high axial resolution and low scattered signal crosstalk, be suitable for deep tissues tomographic spectroscopy micro-imaging；
Spectral information acquisition speed is improved, it can be achieved that wide visual field high speed spectrum micro-imaging.
Wide visual field chromatography ultraphotic spectrum micro imaging method proposed by the invention is described in detail with device above,
Principle and implementation of the present invention are described for specific case used herein, and the explanation of above example is only used
Understand the method and core concept of the present invention in help；Meanwhile for those of ordinary skill in the art, think of according to the present invention
Think, there will be changes in the specific implementation manner and application range, these changes should all belong to the right appended by the present invention
It is required that protection domain.In conclusion the content of the present specification should not be construed as limiting the invention.
Claims (6)
1. a kind of wide visual field chromatography ultraphotic focused based on space-time composes micro imaging method, which is characterized in that include the following steps：
1) parameter setting：Setting lateral, longitudinal direction and axial respectively x-axis, y-axis and z-axis along sample, set along laser spectrum direction
For λ axis, set along time dimension direction as t axis；The target scan areas XYZ in sample is set, setting is realized along sample longitudinal direction
The galvanometer deflection angle step-length of line scanning, the microcobjective axial direction step-length along sample axial scan is realized in setting, according to targeted scans
The size setting spectral information collection period and scanning total duration in region；
2) ultra-short pulse laser light source is utilized to generate ultra-short pulse laser；
3) it in start time scan period, is formed in the sample by space-time focus method simultaneously in space and time two
The focal line that dimension focuses；
4) fluorescence signal is inspired on the focal line of step 3) by nonlinear optical effect, the fluorescence signal is via micro- object
Reverse transfer after mirror is collected, is then filtered out the ultra-short pulse laser of reflection and is filtered out sample by confocal optics slit and dissipate by filter plate
Caused stray light is penetrated, the bar shaped transmitting fluorescence of sample is obtained；
5) obtained bar shaped is emitted by fluorescence by dispersion element and carries out spectral evolution, carrying out spectral information by planar array detector adopts
Collection, obtains (x, λ) two-dimensional signal of sample；Meanwhile the deflection angle scanned according to the galvanometer deflection angle step-size change line of setting, it obtains
(x, λ, y) three-dimensional information of sample is obtained, until the target areas scanning traversal XY, complete sample two dimensional surface different location spectrum and believe
The acquisition of breath；Wherein, the expansion degree of the bar shaped transmitting fluorescence is completely covered in the search coverage size of the planar array detector,
And the planar array detector is synchronous with the trigger signal of galvanometer；
6) according to the depth of the microcobjective axial direction step-size change focal line of setting, (x, λ, y, z) four-dimensional information of sample is obtained,
Until the target areas scanning traversal XYZ, complete the acquisition of different depth spectral information, obtain three dimensions different location in sample
Spectral information；Present scanning cycle terminates, and executes step 7)；
7) step 3)~step 6) is repeated according to the spectral information collection period of setting and composes micro-imaging into line delay ultraphotic, obtained
(x, λ, y, z, t) five-dimensional information of sample, until reach the scanning total duration of setting, complete wide visual field chromatography ultraphotic spectrum it is micro- at
Picture.
2. a kind of device for composing micro imaging method based on the wide visual field chromatography ultraphotic that space-time focuses according to claim 1,
It is characterised in that it includes ultra-short pulse laser light source and beam shaping system, the linear scanning system based on space-time focusing, optics are aobvious
Micro-system and filtering and synchronous spectrum confocal detection system；Wherein,
The ultra-short pulse laser light source and beam shaping system, ultra-short pulse laser light source generate nonlinear optics for providing
The excitation pulse light of signal, beam shaping system is for adjusting the excitation pulse light beam sizes；
It is described based on space-time focus linear scanning system, be placed in after the beam shaping system, including diffractive-optical element, thoroughly
Mirror and optical scanning element, the diffractive-optical element and the optical scanning element are respectively placed in the object space focal plane of the lens
With image space focal plane；The diffractive-optical element is used to introduce the angle dispersion of excitation pulse light beam, and the optical scanning element is used for
Introduce the variable deflection angle of excitation pulse light beam；
The optical microscope system is placed in after the optical scanning element, including lens group and microcobjective, by the lens
Group connects entrance pupil face after the optical scanning element and microcobjective, constitutes 4f systems, which is used in sample
It is middle to form the focal line focused simultaneously in two dimensions in space and time, to excite tissue sample and be based on nonlinear optical effect
Generate transmitting fluorescence；
The filtering and synchronous spectrum confocal detection system, are placed in the linear scanning system focused based on space-time and emit fluorescence
After the simultaneously optical scanning element of reverse transfer being collected through the microcobjective, including filter plate, confocal optics slit and spectrum
Instrument, transmitting fluorescence signal for selecting sample simultaneously carry out spectral information acquisition；The acquisition of information of the spectrometer and the base
It is synchronized in the trigger signal for the linear scanning system that space-time focuses.
3. wide visual field chromatography ultraphotic according to claim 2 composes microscopic imaging device, which is characterized in that the ultrashort pulse
In laser light source and beam shaping system, it is additionally provided with dispersion pre-compensation system before ultra-short pulse laser output, for mending in advance
Repay the dispersion that ultrashort pulse is accumulated before reaching microcobjective focusing surface.
4. wide visual field chromatography ultraphotic according to claim 2 composes microscopic imaging device, which is characterized in that described to be based on space-time
The linear scanning system of focusing technology further includes being placed in Fu of the diffractive-optical element edge excitation optical propagation direction after lens
Adaptive optical element at blade face further overcomes biological sample scattering to being ultimately imaged for carrying out spectrum phase shaping
Caused by influence.
5. wide visual field chromatography ultraphotic according to claim 2 composes microscopic imaging device, which is characterized in that the ultrashort pulse
In laser light source and beam shaping system, ultra-short pulse laser light source selects femtosecond pulse light according to output pulse width
Source or picosecond pulse laser light source；Whether ultra-short pulse laser light source is adjustable according to output wavelength, selects the ultrashort of fixed wave length
The ultra-short pulse laser light source of pulsed laser light source or tunable wavelength；Beam shaping system is Galilean telescope system or opens
General Le telescopic system；
The ultra-short pulse laser light source and beam shaping system provide nonlinear optical in the exciting light for generating non linear optical signal
It learns signal and passes through two-photon absorption fluorescent effect, Three-photon absorption fluorescent effect or two-photon excitation-fluorescence resonance energy transfer
Any one of effect generates.
6. wide visual field according to claim 2 chromatography ultraphotic composes microscopic imaging device, which is characterized in that the filtering with it is same
It walks in Spectral Confocal detection system, spectrometer is made of dispersion element, two-dimensional area detector and two lens；Wherein, described total
Burnt optical slot is placed in the conjugate planes in sample excitation face, and confocal optics slit and dispersion element are constituted image and closed by the first lens
Dispersion element and two-dimensional area detector are constituted object-image relation by system, the second lens.
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| PCT/CN2018/094531 WO2019232875A1 (en) | 2018-06-08 | 2018-07-04 | Spatial and temporal focusing-based wide field of view chromatography hyperspectral microscopic imaging method and device |
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| CN108742532B (en) | 2020-04-24 |
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