CN108742532B - Wide-field chromatographic ultra-spectral microscopic imaging method and device based on space-time focusing - Google Patents
Wide-field chromatographic ultra-spectral microscopic imaging method and device based on space-time focusing Download PDFInfo
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Abstract
The invention discloses a wide-field chromatography hyper-spectral microscopic imaging method and device based on space-time focusing, and belongs to the technical field of microscopic spectral imaging and analytical chemistry. The method comprises the steps of generating ultrashort pulse laser by using an ultrashort pulse laser light source, generating a focusing line in a sample by adopting space-time focusing, collecting excited fluorescence, filtering stray light by adopting a confocal optical slit, collecting fluorescence spectrum information to obtain spectrum information (x, lambda) of the sample, and finally obtaining five-dimensional information of the sample (x, lambda, y, z, t) by three-dimensional space scanning and time-delay scanning. The device comprises an ultrashort pulse laser source, a light beam conversion system, a line scanning system based on space-time focusing, an optical microscope system and a filtering and synchronous spectrum confocal detection system, wherein spectrum information in the filtering and synchronous spectrum confocal detection system is acquired and is synchronous with a line scanning trigger signal in the line scanning system combined with the space-time focusing technology. The invention has the advantages of wide field of view, high spatial resolution, high temporal resolution, high spectral resolution and the like.
Description
Technical Field
The invention relates to a wide-field chromatography hyper-spectral microscopic imaging method and device based on space-time focusing, belonging to the technical field of microscopic spectral imaging and analytical chemistry.
Background
Hyper-spectral microscopic imaging (Hyperspectral Microscopy) has important application in the field of biomedical research, and is increasingly widely regarded by people particularly in the fields of clinical disease diagnosis, intraoperative image navigation and the like. The hyperspectral microimaging technology is adopted to obtain spatially distinguishable spectral information, and information such as physiological parameters, morphology, components and the like of biological tissues can be provided for disease diagnosis. Currently, non-invasive detection of a variety of cancers has been achieved using hyperspectral microscopy imaging techniques.
Essentially, hyperspectral microimaging is a technique for acquiring higher dimensional information (i.e., spectral information) based on microscopic imaging. According to the implementation mode of the microscopic imaging technology, the current hyperspectral microscopic imaging technology can be divided into the hyperspectral microscopic imaging technology based on common wide-field microscopy, the hyperspectral microscopic imaging technology based on confocal scanning and the like. The former can quickly and parallelly acquire spectral information in a wide field of view, but is limited to the defects that a common wide-field microscope does not have the chromatography capability and is easy to cause signal crosstalk by tissue scattering, and the like, and the technology is only suitable for transparent biological samples. The latter is based on the confocal principle, and to a certain extent, suppresses the influence of tissue scattering and obtains axial resolution capability, but because point-by-point scanning imaging needs to be performed, the imaging speed is limited. Furthermore, in recent years, hyper-spectral microscopy imaging techniques based on light sheet microscopy have emerged, which unfortunately are equally unsuitable for diffuse tissue imaging.
In order to overcome the influence of biological tissue scattering and improve the imaging penetration depth, people introduce a nonlinear optical microscopy technology into hyperspectral microscopy imaging, develop a hyperspectral microscopy technology based on a nonlinear optical effect, and widely apply the hyperspectral microscopy technology to biomedical research. Since the common non-linear optical microscopy still adopts a point scanning mode to overcome the influence of tissue scattering, the imaging speed and flux are influenced. On the other hand, the nonlinear optical microscopy adopting surface excitation avoids the speed bottleneck caused by point-to-point scanning, but the excited signal is seriously interfered after being scattered by the tissue, and is not suitable for scattered tissue spectral imaging. The conventional non-linear optical microscopy technique using line scanning can compromise between imaging speed and suppression of the effects of scattering, but this method is not an ideal choice, as opposed to the reduced axial resolution obtained with point scanning.
Therefore, one technical problem that needs to be urgently solved by those skilled in the art is: how to creatively provide an effective measure to solve the defects in the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a wide-field chromatography hyper-spectral microscopic imaging method and device based on space-time focusing. The invention is suitable for scattering biological tissue imaging, can improve imaging speed and flux, and has high axial resolution.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a wide-field chromatography hyper-spectral microscopic imaging method based on space-time focusing, which is characterized by comprising the following steps of:
1) setting parameters: setting an x axis, a y axis and a z axis respectively along the transverse direction, the longitudinal direction and the axial direction of a sample, setting a lambda axis along the laser spectrum direction, and setting a t axis along the time dimension direction; setting a target scanning area XYZ in a sample, setting a galvanometer deflection angle step length for realizing scanning along a longitudinal line of the sample, setting a microscope objective axial step length for realizing axial scanning along the sample, and setting a spectrum information acquisition period and total scanning duration according to the size of the target scanning area;
2) generating ultrashort pulse laser by using an ultrashort pulse laser light source;
3) forming a focusing line which is focused in two dimensions of space and time simultaneously in a sample by a space-time focusing method at the starting moment of a scanning period;
4) exciting a fluorescence signal on the focusing line in the step 3) through a nonlinear optical effect, collecting the fluorescence signal through a microscope objective lens, transmitting the fluorescence signal in a reverse direction, filtering reflected ultrashort pulse laser by a filter plate, and filtering stray light caused by sample scattering by a confocal optical slit to obtain strip-shaped emission fluorescence of the sample;
5) performing spectrum expansion on the obtained bar-shaped emission fluorescence through a dispersion element, and acquiring spectral information through an area array detector to obtain (x, lambda) two-dimensional information of a sample; meanwhile, changing the deflection angle of line scanning according to the set step length of the deflection angle of the galvanometer to obtain (x, lambda, y) three-dimensional information of the sample until the XY target area is traversed by scanning, and acquiring spectral information of different positions of a two-dimensional plane of the sample; the size of a detection area of the area array detector completely covers the unfolding degree of the strip-shaped emission fluorescence, and the area array detector is synchronous with a trigger signal of the galvanometer;
6) changing the depth of a focal line according to the set axial step length of the microscope objective to obtain (x, lambda, y, z) four-dimensional information of the sample until the XYZ target area is scanned and traversed, and finishing the acquisition of spectral information of different depths to obtain spectral information of different positions of a three-dimensional space in the sample; when the current scanning period is finished, executing step 7);
7) and (3) repeating the steps 3) to 6) according to a set spectrum information acquisition period to perform time-delay hyperspectral microimaging, and obtaining (x, lambda, y, z, t) five-dimensional information of the sample until a set total scanning time is reached, thereby completing the wide-field chromatography hyperspectral microimaging.
The invention also provides a device for the wide-field chromatography ultra-spectral microscopic imaging method based on the space-time focusing, which is characterized by comprising an ultra-short pulse laser source, a light beam conversion system, a line scanning system based on the space-time focusing, an optical microscopic system and a filtering and synchronous spectrum confocal detection system; wherein,
the ultra-short pulse laser light source is used for providing excitation pulse light for generating nonlinear optical signals, and the light beam conversion system is used for adjusting the size of the excitation pulse light beam;
the line scanning system based on the space-time focusing is arranged behind the light beam transformation system and comprises an optical diffraction element, a lens and an optical scanning element, wherein the optical diffraction element and the optical scanning element are respectively arranged on an object focal plane and an image focal plane of the lens; the optical diffraction element is used for introducing the angular dispersion of the excitation pulse beam, and the optical scanning element is used for introducing the variable deflection angle of the excitation pulse beam;
the optical microscope system is arranged behind the optical scanning element, comprises a lens group and a microscope objective, and is connected with the optical scanning element and the microscope objective through the lens group to form a 4f system, wherein the optical microscope system is used for forming a focal line which focuses in two dimensions of space and time simultaneously in a sample so as to excite the tissue sample and generate emission fluorescence based on a nonlinear optical effect;
the filtering and synchronous spectrum confocal detection system is arranged in the space-time focusing-based line scanning system and is used for selecting a fluorescence emission signal of a sample and acquiring spectrum information, and comprises a filter, a confocal optical slit and a spectrometer, wherein the optical scanning element is used for collecting and reversely transmitting fluorescence emitted by the line scanning system through the microscope objective; the information acquisition of the spectrometer is synchronized with the trigger signal of the spatio-temporal focusing based line scanning system.
Compared with the prior art, the invention has the following advantages: by adopting a line scanning technology based on a space-time focusing method and providing a corresponding synchronous spectrum confocal detection technology, high axial resolution, low scattering signal crosstalk and high-speed spectrum information acquisition can be ensured, and the realized wide-field chromatography hyper-spectral micro-imaging technology is suitable for deep tissue wide-field high-speed chromatography micro-spectral imaging.
In summary, the proposed wide-field chromatographic hyperspectral microimaging technology can be used for acquiring (x, y, z, t, lambda) five-dimensional information of deep biological tissues, and has the advantages of wide field, high spatial resolution, high temporal resolution, high spectral resolution and the like.
Drawings
FIG. 1 is a schematic structural diagram of a wide-field chromatography hyper-spectral microscopic imaging device according to the invention.
Fig. 2 is a schematic diagram of the principle of the present invention.
FIG. 3 is a schematic diagram of the structure of an embodiment 1 of the apparatus of the present invention.
FIG. 4 is a schematic diagram of the structure of an embodiment 2 of the apparatus of the present invention.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
The invention provides a wide-field chromatography hyper-spectral microscopic imaging method, which specifically comprises the following steps:
1) setting parameters: setting the X axis, the Y axis and the Z axis respectively along the transverse direction, the longitudinal direction and the axial direction of a sample, setting the X axis along the laser spectrum (namely wavelength) direction, and setting the t axis along the time dimension direction; setting a target scanning area XYZ in a sample, setting a galvanometer deflection angle step length for realizing scanning along a longitudinal line of the sample, setting a microscope objective axial step length for realizing axial scanning along the sample, and setting a spectrum information acquisition period and total scanning duration according to the size of the target scanning area;
2) generating ultrashort pulse laser by using an ultrashort pulse laser light source;
3) at the beginning of a scanning period, forming a focal line which focuses in two dimensions of space and time simultaneously in a sample by a space-time focusing method (along the x direction, the y-direction scanning of the focal line can be realized by changing the deflection angle of a vibrating mirror);
4) exciting a fluorescence signal on the focusing line in the step 3) through a nonlinear optical effect, collecting the fluorescence signal through a microscope objective lens, transmitting the fluorescence signal in a reverse direction, filtering reflected ultrashort pulse laser by a filter plate, and filtering stray light caused by sample scattering by a confocal optical slit to obtain strip-shaped emission fluorescence of the sample;
5) performing spectrum expansion on the obtained bar-shaped emission fluorescence through a dispersion element, and acquiring spectral information through an area array detector to obtain (x, lambda) two-dimensional information of a sample; meanwhile, changing the deflection angle of line scanning according to the set step length of the deflection angle of the galvanometer to obtain (x, lambda, y) three-dimensional information of the sample until the XY target area is traversed by scanning, and acquiring spectral information of different positions of a two-dimensional plane of the sample; the size of a detection area of the area array detector completely covers the unfolding degree of the strip-shaped emitted fluorescence, and the area array detector is synchronous with a trigger signal of the galvanometer;
6) changing the depth of a focal line according to the set axial step length of the microscope objective to obtain (x, lambda, y, z) four-dimensional information of the sample until the XYZ target area is scanned and traversed, and finishing the acquisition of spectral information of different depths to obtain spectral information of different positions of a three-dimensional space in the sample; when the current scanning period is finished, executing step 7);
7) and (3) repeating the steps 3) to 6) according to a set spectrum information acquisition period to perform time-delay hyperspectral microimaging, and obtaining (x, lambda, y, z, t) five-dimensional information of the sample until a set total scanning time is reached, thereby completing the wide-field chromatography hyperspectral microimaging.
The invention also provides a wide-field chromatography hyper-spectral microscopic imaging device according to the method, the structure of which is shown in figure 1, and the device comprises: the system comprises an ultra-short pulse laser light source, a light beam conversion system, a line scanning system based on space-time focusing, an optical microscope system and a filtering and synchronous spectrum confocal detection system; wherein,
the system comprises an ultra-short pulse laser light source and a light beam conversion system, wherein the ultra-short pulse laser light source is used for providing excitation pulse light for generating nonlinear optical signals, and the light beam conversion system is used for adjusting the size of the excitation pulse light beam;
the line scanning system based on space-time focusing is arranged behind the light beam transformation system and comprises an optical diffraction element, a lens and an optical scanning element, wherein the optical diffraction element and the optical scanning element are respectively arranged on an object focal plane and an image focal plane of the lens; the optical diffraction element is used for introducing the angular dispersion of the excitation pulse beam, and the optical scanning element is used for introducing the variable deflection angle of the excitation pulse beam;
the optical microscope system is arranged behind the optical scanning element, comprises a lens group and a microscope objective, is connected with the optical scanning element and the microscope objective through the lens group, and forms a 4f system, and is used for forming a focusing line which focuses in two dimensions of space and time simultaneously in a sample so as to excite a tissue sample and generate emission fluorescence based on a nonlinear optical effect;
the filtering and synchronous spectrum confocal detection system is arranged in a space-time focusing-based line scanning system, is used for transmitting fluorescence, is collected by a microscope objective and is reversely transmitted by an optical scanning element, comprises a filter, a confocal optical slit and a spectrometer, and is used for selecting a fluorescence signal transmitted by a sample and collecting spectrum information; wherein the information acquisition of the spectrometer is synchronized with a trigger signal of the line scanning system based on spatio-temporal focusing.
Furthermore, in the ultra-short pulse laser light source and the light beam conversion system, a dispersion pre-compensation system is arranged before the ultra-short pulse laser is output, and is used for pre-compensating the dispersion accumulated before the ultra-short pulse reaches the focusing surface of the microscope objective.
Furthermore, the line scanning system based on the space-time focusing technology also comprises an adaptive optical element which is arranged on the Fourier surface of the optical diffraction element after passing through the lens along the propagation direction of the exciting light and is used for performing spectral phase shaping and further overcoming the influence of the scattering of the biological sample on the final imaging.
The specific implementation manner of each component in the device of the invention is as follows:
in the ultrashort pulse laser source and the light beam conversion system, the ultrashort pulse laser source can select a femtosecond pulse laser source or a picosecond pulse laser source according to the output pulse width; the ultrashort pulse laser source can select an ultrashort pulse laser source with fixed wavelength or an ultrashort pulse laser source with tunable wavelength according to whether the output wavelength is tunable or not; the light beam transformation system is a Galileo telescope system or a Keplerian telescope system. The ultrashort pulse laser source and the light beam conversion system provide the nonlinear optical signal in the exciting light for generating the nonlinear optical signal, and the nonlinear optical signal is generated through any one of a two-photon absorption fluorescence effect, a three-photon absorption fluorescence effect or a two-photon excitation-fluorescence resonance energy transfer effect.
In the line scanning system based on the space-time focusing technology, the optical diffraction element can be selected from a grating, a deformable mirror, a spatial light modulator or other optical diffraction elements; the optical scanning element is selected from a galvanometer, a polygonal mirror, an acousto-optic modulator and the like.
In the filtering and synchronous spectrum confocal detection system, the filter plate is selected from a dichroic mirror, a band-pass filter plate, a low-pass filter plate or a long-pass filter plate. The confocal optical slit is arranged on the conjugate plane of the excitation plane of the biological sample, and the width of the confocal optical slit is determined by the design size of the conjugate image of the sample. The spectrometer consists of a dispersion element, a two-dimensional plane detector and two lenses, wherein the first lens enables a confocal optical slit and the dispersion element to form an object-image relationship, and the second lens enables the dispersion element and the two-dimensional plane detector to form the object-image relationship; the dispersive element can be a prism, a grating or other dispersive elements; the two-dimensional surface detector is selected from a Charge Coupled Device (CCD), an Electron Multiplying Charge Coupled Device (EMCCD) or a scientific grade complementary metal oxide semiconductor device (sCMOS) and the like.
Referring to fig. 2, a schematic diagram of the principles of the present invention is shown. By using nonlinear optical effect and combining with space-time focusing technology (U.S. patent No. 20080151238a1), a focusing line (x direction) with high axial resolution can be generated on a biological sample, and an excited fluorescence spectrum signal is subjected to confocal detection to be imaged on a two-dimensional photoelectric detection surface (Dx, Dy) of a spectrometer, wherein the Dy direction is a spectrum lambda dimension. By scanning in the y direction of the sample and performing synchronous confocal detection, three-dimensional (x, lambda, y) information of the sample under a wide field of view can be obtained. By using the axial tomography capability of the technology to carry out axial scanning (namely, changing the relative position of the moving sample and the microscope objective), the (x, lambda, y, z) four-dimensional information of the sample can be obtained; further, by utilizing the high-speed spectral microscopic imaging capability of the technology to perform time-delay information acquisition (namely acquiring four-dimensional information of the sample (x, lambda, y, z) at different moments), five-dimensional information (x, lambda, y, z, t) of the sample can be obtained. Therefore, the wide-field chromatography hyper-spectral microscopic imaging method and the device thereof have the advantages of wide field, high spatial resolution, high temporal resolution, high spectral resolution and the like, and can provide rich information for biodynamic process research, disease diagnosis basis and the like.
Example 1:
referring to fig. 3, the wide-field tomography hyperspectral microimaging apparatus of the embodiment is described in detail, and the apparatus includes an ultrashort pulse laser source and beam transformation system, a line scanning system based on space-time focusing, an optical microscopy system, and a filtering and synchronous spectrum confocal detection system, and a biological sample is placed on a sample stage 319. Wherein, the ultrashort pulse laser source 301 in the ultrashort pulse laser source and beam transformation system adopts a femtosecond laser (such as Coherent Chameleon Discovery series), and the beam transformation system adopts a keplerian telescope system (4 f system) composed of a lens 302 and a cylindrical lens 303; the line scanning system based on space-time focusing comprises a transmission grating 304, a lens 305 and a scanning galvanometer 307; the optical microscope system comprises two lenses 308, 309 and a microscope objective 310; the filtering and synchronous spectrum confocal detection system comprises a dichroic mirror 306, a low-pass filter 311, a lens 312, a confocal optical slit 313 (the width of the optical slit is determined by the design size of a conjugate image of a biological sample), and a spectrometer consisting of two lenses 314 and 316, a reflection grating 315 and a two-dimensional surface detector (adopting sCMOS or EMCCD) 317. The relative position relation of the components is as follows: the lens 302 and the cylindrical lens 303 form a 4f system for expanding beams, the transmission grating 304 is arranged at an image surface of the cylindrical lens 303, the lens 305 images the transmission grating 304 at the scanning galvanometer 307, the lenses 308 and 309 form a 4f system so that the scanning galvanometer 307 is conjugated with an entrance pupil surface of the microscope objective 310, the lens 312 and the lens 308 form a 4f system so that an object surface is imaged at a confocal optical slit 313, the low-pass filter 311 is arranged in front of the lens 312, the lens 314 images the confocal optical slit 313 at the reflection grating 315, and the lens 316 images the reflection grating 315 at the two-dimensional plane detector 317. Also illustrated in fig. 3 is a computer 318 for controlling the deflection angle of the scanning galvanometer 307 and performing conventional image reconstruction and data processing on the spectral information collected by the two-dimensional area detector 317.
In this embodiment, a laser beam emitted from the ultra-short pulse laser source 301 is expanded by the lens 302 and the cylindrical lens 303 (diameter of the laser beam is changed), and then enters the transmission grating 304, the ultra-short pulse light beam generates angular dispersion under the action of the transmission grating 304 (in order to make the light beam introduced with the angular dispersion fill the back focal plane of the objective lens after passing through a subsequent optical element), is collimated by the lens 305, then is projected onto the scanning galvanometer 307 through the dichroic mirror 306, is introduced with a variable deflection angle (the deflection angle is driven by galvanometer driving voltage, and a deflection angle is set according to a scanning area), and finally generates a focal line on the focal plane of the objective lens in the biological sample through the lenses 308, 309 and the microscope objective lens 310. The optical signal generated by the nonlinear optical effect is collected by the microscope objective 310 and then transmitted in reverse direction, passes through the lenses 309 and 308 and the scanning galvanometer 307 in sequence, and is reflected by the dichroic mirror 306. Then, the signal beam sequentially passes through the low pass filter 311, the lens 312 and the confocal optical slit 313, and finally enters the spectrometer for signal collection. It is noted that the line scan trigger signal of the scanning galvanometer 307 is synchronized with the frame trigger signal of the two-dimensional area detector 317. By adopting the technical scheme, the (x, lambda, y) three-dimensional information of the biological sample can be obtained. By moving the microscope objective 310 for axial scanning, (x, λ, y, z) four-dimensional information of the sample can be obtained. If the time delay information acquisition is carried out, five-dimensional (x, lambda, y, z, t) information of the sample can be obtained.
Example 2:
referring to fig. 4, the wide-field tomosynthesis hyperspectral microscopy imaging apparatus of the present embodiment will be described in detail, and the present embodiment is different from embodiment 1 in that an adaptive optical element is added. The device comprises an ultrashort pulse laser source, a light beam conversion system, a line scanning system based on space-time focusing, an optical microscope system and a filtering and synchronous spectrum confocal detection system, wherein a biological sample is placed on a sample stage 424; wherein, the ultrashort pulse laser source 401 in the ultrashort pulse laser source and beam transformation system adopts a femtosecond laser (such as Coherent Chameleon Discovery series), and the beam transformation system adopts a keplerian telescope system (4 f system) composed of a lens 402 and a cylindrical lens 403; the line scanning system based on space-time focusing comprises a transmission grating 404, five lenses 405, 407, 408, 410 and 411, an adaptive optical element 406 (a spatial light modulator is adopted in the embodiment) arranged between the lenses 405 and 407, and a scanning galvanometer 412; the optical microscope system comprises two lenses 413, 414, a microscope objective 415; the filtering and synchronous spectrum confocal detection system comprises a dichroic mirror 409, a low-pass filter 416, a common lens 417, a confocal optical slit 418 (the width of the optical slit is determined by the design size of a conjugate image of a biological sample), and a spectrometer consisting of two lenses 419 and 421, a reflection grating 420 and a two-dimensional surface detector (adopting sCMOS or EMCCD) 422. The relative position relation of the components is as follows: the lens 402 and the cylindrical lens 403 form a 4f system for beam expansion, the transmission grating 404 is placed at the image surface of the cylindrical lens 403, the lens 405 images the transmission grating 404 at the spatial light modulator 406, the lenses 407 and 408, the lenses 410 and 411, and the lenses 413 and 414 respectively form 3 groups of series-connected 4f systems, so that the spatial light modulator 406 is conjugate with the entrance pupil surface of the scanning galvanometer 412 and the microscope objective 415, the lenses 417 and 410 form a 4f system, so that the object surface is imaged at the confocal optical slit 418, the low-pass filter 416 is placed immediately before the lens 417, the lens 419 images the confocal optical slit 418 at the reflection grating 420, and the lens 421 images the reflection grating 420 at the two-dimensional plane detector 422. Fig. 4 also illustrates a computer 423 for controlling the deflection angle of the scanning galvanometer 412 and performing conventional image reconstruction and data processing on the spectral information collected by the two-dimensional detector 422.
In this embodiment, a laser beam emitted by an ultrashort pulse laser source 401 is expanded by a lens 402 and a cylindrical lens 403 and then enters a transmission grating 404, the ultrashort pulse beam generates angular dispersion under the action of the transmission grating 404, is collimated by a lens 405 and then is projected to a spatial light modulator 406 for spectral phase shaping so as to further overcome the influence of tissue scattering, then sequentially passes through a lens 407, a lens 408, a dichroic mirror 409, a lens 410 and a lens 411 to introduce a variable deflection angle on a scanning galvanometer 412, and finally generates a focal line on an objective lens focal plane in a biological sample through the lenses 413 and 414 and a microscope objective lens 415. The fluorescence signal generated by the nonlinear optical effect is collected by the microscope objective 415 and then transmitted in reverse direction, passes through the lens 414, the lens 413, the scanning galvanometer 412, the lens 411 and the lens 410 in sequence, and is reflected by the dichroic mirror 409. Then, the signal beam sequentially passes through the low pass filter 416, the lens 417 and the confocal optical slit 418, and finally enters the spectrometer for signal collection. It should be noted that the line scanning trigger signal of the scanning galvanometer 412 is synchronized with the frame trigger signal of the two-dimensional surface detector 422; spatial light modulator 406 measures wavefront distortion using adaptive optics and applies a compensating wavefront (prior to spectral imaging). By adopting the technical scheme, the (x, lambda, y) three-dimensional information of the biological sample can be obtained. By moving microscope objective 415 for axial scanning, (x, λ, y, z) four-dimensional information of the biological sample can be obtained. If time-lapse information acquisition (i.e., multiple acquisition) is performed, five-dimensional (x, λ, y, z, t) information of the biological sample can be obtained.
In practical experiments, considering that the required excitation light power may be larger than the light damage threshold of the spatial light modulator, it may also be necessary to add a lens group before the spatial light modulator 406 so that the light beam spreads in a direction perpendicular to the spectral spread to a light intensity below the light damage threshold of the spatial light modulator.
In conclusion, the line scanning technology is carried out by combining the time-space focusing technology, and the corresponding synchronous spectrum confocal detection technology is provided, so that the high axial resolution and the low scattering signal crosstalk are ensured, and the method is suitable for deep tissue chromatographic microscopic imaging; the acquisition speed of spectral information is improved, and wide-field high-speed spectral microscopic imaging can be realized.
The method and the device for wide-field chromatography hyperspectral microscopy imaging provided by the invention are described in detail, specific examples are applied in the method to explain the principle and the implementation mode of the invention, and the description of the examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there are changes in the specific embodiments and the application scope, and these changes should be covered by the protection scope of the appended claims. In view of the above, the present disclosure should not be construed as limiting the invention.
Claims (6)
1. A wide-field chromatography hyper-spectral microscopic imaging method based on space-time focusing is characterized by comprising the following steps:
1) setting parameters: setting an x axis, a y axis and a z axis respectively along the transverse direction, the longitudinal direction and the axial direction of a sample, setting a lambda axis along the laser spectrum direction, and setting a t axis along the time dimension direction; setting a target scanning area XYZ in a sample, setting a galvanometer deflection angle step length for realizing scanning along a longitudinal line of the sample, setting a microscope objective axial step length for realizing axial scanning along the sample, and setting a spectrum information acquisition period and total scanning duration according to the size of the target scanning area;
2) generating ultrashort pulse laser by using an ultrashort pulse laser light source;
3) forming a focusing line which is focused in two dimensions of space and time simultaneously in a sample by a space-time focusing method at the starting moment of a scanning period;
4) exciting a fluorescence signal on the focusing line in the step 3) through a nonlinear optical effect, collecting the fluorescence signal through a microscope objective lens, transmitting the fluorescence signal in a reverse direction, filtering reflected ultrashort pulse laser by a filter plate, and filtering stray light caused by sample scattering by a confocal optical slit to obtain strip-shaped emission fluorescence of the sample;
5) performing spectrum expansion on the obtained bar-shaped emission fluorescence through a dispersion element, and acquiring spectral information through an area array detector to obtain (x, lambda) two-dimensional information of a sample; meanwhile, changing the deflection angle of line scanning according to the set step length of the deflection angle of the galvanometer to obtain (x, lambda, y) three-dimensional information of the sample until the XY target area is traversed by scanning, and acquiring spectral information of different positions of a two-dimensional plane of the sample; the size of a detection area of the area array detector completely covers the unfolding degree of the strip-shaped emission fluorescence, and the area array detector is synchronous with a trigger signal of the galvanometer;
6) changing the depth of a focal line according to the set axial step length of the microscope objective to obtain (x, lambda, y, z) four-dimensional information of the sample until the XYZ target area is scanned and traversed, and finishing the acquisition of spectral information of different depths to obtain spectral information of different positions of a three-dimensional space in the sample; when the current scanning period is finished, executing step 7);
7) and (3) repeating the steps 3) to 6) according to a set spectrum information acquisition period to perform time-delay hyperspectral microimaging, and obtaining (x, lambda, y, z, t) five-dimensional information of the sample until a set total scanning time is reached, thereby completing the wide-field chromatography hyperspectral microimaging.
2. The device for the wide-field chromatographic ultra-spectral microscopic imaging method based on the space-time focusing is characterized by comprising an ultra-short pulse laser light source and a light beam transformation system, a line scanning system based on the space-time focusing, an optical microscopic system and a filtering and synchronous spectral confocal detection system; wherein,
the ultra-short pulse laser light source is used for providing excitation pulse light for generating nonlinear optical signals, and the light beam conversion system is used for adjusting the size of the excitation pulse light beam;
the line scanning system based on the space-time focusing is arranged behind the light beam transformation system and comprises an optical diffraction element, a lens and an optical scanning element, wherein the optical diffraction element and the optical scanning element are respectively arranged on an object focal plane and an image focal plane of the lens; the optical diffraction element is used for introducing the angular dispersion of the excitation pulse beam, and the optical scanning element is used for introducing the variable deflection angle of the excitation pulse beam;
the optical microscope system is arranged behind the optical scanning element, comprises a lens group and a microscope objective, and is connected with the optical scanning element and the microscope objective through the lens group to form a 4f system, wherein the optical microscope system is used for forming a focal line which focuses in two dimensions of space and time simultaneously in a sample so as to excite the tissue sample and generate emission fluorescence based on a nonlinear optical effect;
the filtering and synchronous spectrum confocal detection system is arranged in the space-time focusing-based line scanning system and is used for selecting a fluorescence emission signal of a sample and acquiring spectrum information, and comprises a filter, a confocal optical slit and a spectrometer, wherein the optical scanning element is used for collecting and reversely transmitting fluorescence emitted by the line scanning system through the microscope objective; the information acquisition of the spectrometer is synchronized with the trigger signal of the spatio-temporal focusing based line scanning system.
3. The apparatus of claim 2, wherein a dispersion pre-compensation system is further disposed in the ultra-short pulse laser source and the beam transformation system before the ultra-short pulse laser output for pre-compensating the dispersion accumulated by the ultra-short pulse before reaching the focusing surface of the microscope objective.
4. The apparatus of claim 2, wherein the line scanning system based on spatiotemporal focusing technique further comprises an adaptive optical element disposed at the fourier plane of the optical diffraction element after passing through the lens along the propagation direction of the excitation light for performing spectral phase shaping to further overcome the effect of scattering of the biological sample on the final imaging.
5. The device of the wide-field chromatography hyper-spectral microscopic imaging method based on the space-time focusing as claimed in claim 2, wherein in the ultra-short pulse laser light source and the light beam transformation system, the ultra-short pulse laser light source selects a femtosecond pulse laser light source or a picosecond pulse laser light source according to the output pulse width; selecting an ultrashort pulse laser source with fixed wavelength or an ultrashort pulse laser source with tunable wavelength according to whether the output wavelength is tunable or not; the light beam transformation system is a Galileo telescope system or a Keplerian telescope system;
the ultrashort pulse laser source and the light beam conversion system provide the nonlinear optical signal in the exciting light for generating the nonlinear optical signal, and the nonlinear optical signal is generated through any one of a two-photon absorption fluorescence effect, a three-photon absorption fluorescence effect or a two-photon excitation-fluorescence resonance energy transfer effect.
6. The apparatus of the wide-field chromatographic ultra-spectral microscopic imaging method based on space-time focusing according to claim 2, wherein in the filtering and synchronous spectrum confocal detection system, the spectrometer consists of a dispersion element, a two-dimensional plane detector and two lenses; the confocal optical slit is arranged on a conjugate surface of a sample excitation surface, the first lens enables the confocal optical slit and the dispersion element to form an object-image relationship, and the second lens enables the dispersion element and the two-dimensional surface detector to form the object-image relationship.
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