CN114153061A - Splicing method with adjustable axial intensity of exciting light based on light sheet imaging - Google Patents
Splicing method with adjustable axial intensity of exciting light based on light sheet imaging Download PDFInfo
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- G02B21/00—Microscopes
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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Abstract
The invention discloses a splicing method with adjustable axial intensity of exciting light based on optical sheet imaging, which comprises the following steps: shaping and filtering light beams emitted by a light source into light beams with preset parameters, and loading Fresnel lens phases with different focal lengths by using the light beams to perform phase modulation; the light beam forms a light sheet through the scanning galvanometer and is used for exciting the sample to generate a fluorescence signal; the light intensity change in the process of transmitting the exciting light is collected and analyzed through the camera, the phase area ratio of Fresnel lenses with different focal lengths loaded on the spatial light modulator is changed, the axial intensity modulation of the exciting light is realized, and the problem of light intensity attenuation caused by absorption in the depth of a sample is compensated. The invention reduces the energy attenuation of the light sheet in the sample transmission process, improves the spatial resolution of the imaging in the sample depth, reduces the image background noise and improves the contrast.
Description
Technical Field
The invention relates to the technical field of biomedical microscopic imaging, in particular to an exciting light axial intensity adjustable splicing method based on optical sheet imaging.
Background
The Light Sheet Fluorescence microscopic imaging technology (LSFM) is a new technology that considers resolution and imaging depth at present and is closest to the ideal target for optical microscope performance optimization, and the technology uses a laser Light Sheet with a certain thickness to excite a contrast agent inside a sample to emit Light, and then collects a Fluorescence signal in a direction perpendicular to the plane of the Light Sheet. Compared with the traditional microscopic imaging means, the optical sheet imaging has the excellent characteristics of high spatial resolution, large visual field, rapid three-dimensional imaging and the like, and has great application potential in the research fields of multiple life sciences such as brain science, organoid and the like.
Over the past decade, light sheet illumination imaging has been rapidly developed and is continuously combined with other advanced optical imaging concepts. The field of light sheet imaging needs a thin light sheet to ensure the axial resolution of imaging and a long light sheet to ensure the field of view of imaging, but the two have contradiction. Among the many methods of producing light sheets, the gaussian light sheet produced by a cylindrical lens is the simplest, but the cross-sectional radius of the gaussian beam is hyperbolic, with the rayleigh length being very short, meaning that the area whose thickness remains relatively thin is very limited. Thereafter, as scanning galvanometers are introduced into the field of light sheet imaging, more and more light sheet microsystems scan incident light beams through an f-theta lens to generate an excitation light sheet, which can more easily combine non-diffracted light beams such as Bessel light beams, Airy light beams and light sheet imaging, and at the same time, the light sheet is generated by dynamic scanning in a mode that makes illumination more uniform.
Although the axial resolution and field of view of a light sheet can be optimized using a scanned, non-diffracted beam, the contradiction between the two still exists. With the continuous optimization of the excitation light path, the adjustable lens system is additionally arranged at the excitation end to move the focus position of the Gaussian beam along the propagation direction of the beam, so that the spliced light sheet is formed and becomes a feasible technical means. At present, methods for producing an axial spliced optical sheet include generating an axial scanning beam focus by using a variable lens system, generating a beam by changing the axial focus position by using a multilayer beam splitter, generating a beam by axially focusing the beam at different positions by phase modulation of a spatial light modulator, and the like.
Although the field of view is effectively expanded by using the methods, in the actual biological tissue imaging process, a sample is mostly a high-absorption medium composed of water, fat and the like, and has a strong absorption effect on light, which causes the problems that the energy of the light sheet is seriously attenuated in the sample propagation process, and finally the spatial resolution of imaging in the deep part of the sample is low, the background noise of the image is high, the contrast is poor and the like, so the current light sheet imaging field still faces the problem that the light intensity attenuation of the exciting light in the absorption medium cannot be compensated.
On the other hand, simply relying on increasing the excitation light power to compensate for absorption by the medium can cause additional optical damage to the tissue bed, and the light intensity cannot be redistributed.
Disclosure of Invention
The invention provides an exciting light axial intensity adjustable splicing method based on light sheet imaging, which solves the problem that the light intensity attenuation of exciting light in an absorption medium cannot be compensated in the existing light sheet imaging field, reduces the energy attenuation of a light sheet in a sample transmission process, improves the spatial resolution of deep imaging of a sample, reduces the background noise of an image, improves the contrast and is described in detail as follows:
an excitation light axial intensity adjustable splicing method based on optical sheet imaging, comprising the following steps:
shaping and filtering light beams emitted by a light source into light beams with preset parameters, and loading Fresnel lens phases with different focal lengths by using the light beams to perform phase modulation;
the light beam forms a light sheet through the scanning galvanometer and is used for exciting the sample to generate a fluorescence signal;
the light intensity change in the process of transmitting the exciting light is collected and analyzed through the camera, the phase area ratio of Fresnel lenses with different focal lengths loaded on the spatial light modulator is changed, the axial intensity modulation of the exciting light is realized, and the problem of light intensity attenuation caused by absorption in the depth of a sample is compensated.
Wherein the light beam with preset parameters is generated before entering the spatial light modulator or is generated by modulation on the spatial light modulator,
the spatial light modulator simultaneously completes the tasks of generating a preset light beam and loading the phase of the Fresnel lens in different areas through phase modulation, finally the spatial light modulator generates spliced complex light beams with adjustable axial intensity, and the spliced complex light beams are scanned by a galvanometer to form light sheets.
Further, the method further comprises: the circular area modulated by the spatial light modulator is subdivided into dense sector areas, and the period of phase change is determined according to the number of focuses.
In one embodiment, the light beam is generated by a spatial light modulator phase, and the final phase is a sector area Fresnel lens phase in a polar periodic form;
and the area distribution of different fan-shaped areas corresponds to the light intensity distribution at different focuses, and a light intensity attenuation image of the laser sheet transmitted in the sample is obtained by primary imaging of the light sheet with preset light intensity uniformly distributed along the axial direction.
Wherein the light beams of the preset parameters are: the spatial light modulator comprises a Gaussian beam, a Bessel beam, an Airy beam and a rotating Airy beam, wherein the beams generated by different fan-shaped areas of the spatial light modulator are focused at different axial positions, and Fresnel lens phases with different focal lengths are loaded in different areas, so that the splicing of different beams is realized.
In one embodiment, the method obtains light intensity attenuation parameters of the light sheet along the propagation direction according to the imaging result, finds out the axial attenuation ratio of the light intensity images in different areas, fits an exponential attenuation curve of the light intensity in the biological sample medium according to the Lambert beer law, and constructs a light intensity attenuation model aiming at the current biological sample.
Furthermore, the method determines the compensated light intensity ratio at different axial focus positions according to the attenuation parameters of different sample models, redistributes dense sector areas on the spatial light modulator, adjusts the sector areas occupied by different focuses according to the energy ratio required by different focuses, and can control the axial light intensity distribution of the spliced light sheet.
The technical scheme provided by the invention has the beneficial effects that:
1) the method provided by the invention has strong applicability, and complex light beams such as Bessel light beams and Airy light beams can be axially spliced besides the traditional Gaussian light beams;
2) the method provided by the invention can match the attenuation influence of different biological tissue samples on the polished section, and can meet different imaging requirements of different biological samples; on the other hand, the invention actually redistributes the input light intensity along the axial direction without causing additional light damage to the tissue superficial layer, unlike simply relying on increasing the excitation light power to compensate for the absorption of the medium;
3) the method provided by the invention is simple to operate, and can be realized by adjusting the patterns loaded on the liquid crystal panel of the spatial light modulator according to the attenuation information of different axial positions in the sample obtained by uniformly splicing the light sheets;
4) the phase pattern finally loaded on the spatial light modulator is periodically arranged in the sector area corresponding to the number of the focuses, so that the input original light beam is uniformly distributed and modulated to generate an axial spliced light beam;
5) the method is not in conflict with other functions of the spatial light modulator, only the Fresnel lens with different focal lengths in different regions is needed to be superposed in the existing phase modulation region, for example, the spatial light modulator can still be used for generating modulated light beams, superposing blazed gratings and the like, and the method is not in conflict with the method for splicing the optical sheet imaging with adjustable excitation light axial intensity.
The splicing method with adjustable axial strength provided by the invention solves the problems and simultaneously realizes further application in the field of light sheet microscopic imaging.
Drawings
FIG. 1 is a schematic structural diagram of an implementation device for excitation light axial intensity adjustable splicing based on optical sheet imaging according to the present invention;
FIG. 2 is a flowchart of a splicing method with adjustable axial intensity of excitation light based on optical sheet imaging according to the present invention;
FIG. 3 is a schematic diagram of the loading phase and the xz direction of the generated scanning beam segment in the splicing method according to the present invention;
FIG. 4 is a schematic diagram of an XZ-direction result of fluorescent particle imaging of an intensity compensation sheet generated by the splicing method provided by the invention.
In the drawings, the components represented by the respective reference numerals are listed below:
1: a light source; 2: a first lambda/2 slide;
3: a polarizing beam splitter; 4: a diaphragm;
5: a second lambda/2 wave plate; 6: a first lens;
7: a pinhole; 8: a second lens;
9: a spatial light modulator; 10: a third lens;
11: a fourth lens; 12: a galvanometer;
13: a fifth lens; 14: a sixth lens;
15: a reflective mirror; 16: a seventh lens;
17: an eighth lens; 18: an illumination objective lens;
19: an imaging objective lens; 20: a cylindrical mirror;
21: an optical filter; 22: a camera.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in further detail below.
Aiming at the problem that the optical sheet imaging technology is applied to the actual biological tissue imaging process, namely, most biological samples are high-absorption media consisting of water, fat and the like, and have strong absorption effect on light, so that the energy of an optical sheet is seriously attenuated in the sample propagation process.
The embodiment of the invention provides an exciting light axial intensity adjustable splicing method based on light sheet imaging, which can fully utilize a currently formed light path, basically does not change the setting of an original light path, only needs to add a spatial light modulator in the light path to perform phase modulation on an input light beam, is not limited to a Gaussian light beam, can use an optical element to generate various types of light beams, and is suitable for the exciting light axial intensity adjustable splicing method provided by the invention.
Example 1
An excitation light axial intensity adjustable splicing method based on light sheet imaging, referring to fig. 1-4, comprises the following steps:
101: shaping and filtering a light beam emitted by a light source into a light beam with preset parameters;
namely, a light beam with preset parameters can be generated by shaping devices such as a laser beam expanding system, a spatial filtering system and the like.
In practical application, the light beams with preset parameters can be generated before entering the spatial light modulator, and can also be generated by modulation on the spatial light modulator, at the moment, the spatial light modulator can simultaneously complete the tasks of generating the preset light beams and loading the phase of the Fresnel lens in different areas through phase modulation, and finally, the spatial light modulator can completely generate spliced complex light beams with adjustable axial intensity, and the spliced complex light beams are scanned by a galvanometer to form light sheets. The circular area modulated by the spatial light modulator is subdivided into dense sector areas, and the period of phase change is determined according to the required number of focuses.
102: the phase modulation is carried out by loading Fresnel lens phases with different focal lengths by utilizing light beams;
the Fresnel lens phases with different focal lengths in the subareas are loaded, and the lasers in different modulation areas are focused at different positions along the axial direction, so that laser splicing is realized. When the split-type optical lens works, the modulated spliced light beam is shaped again through the optical system, an optical slice is formed through scanning of the galvanometer, the galvanometer processes the front focus of a subsequent optical path lens to form an f-theta galvanometer system, a uniform light sheet is generated, and meanwhile, the split-type optical lens only changes the incident angle of the light beam through vibration on the input plane of the objective lens.
The phase of the superposed Fresnel lens is actually equal to that of a lens optical element, the phase of the Fresnel lens introduced with different focal lengths is equal to that of a light beam which is normally transmitted, and the light beam is converged by a convex lens to be focused before an original focus or is diverged by a concave lens to be focused after the original focus. And deducing a preset focal length parameter f and an offset delta x to form an inverse proportional function relation according to a formula of the phase of the Fresnel lens and the focal length of the Fresnel lens, and verifying the offset delta x corresponding to the parameter f through experiments.
103: the light beam forms a light sheet through the scanning galvanometer and is used for exciting the sample to generate a fluorescence signal;
the spliced light beams are scanned to form light sheets, and the light sheets are incident from one side of the sample to excite the sample to generate fluorescence.
104: the light intensity change in the process of transmitting the exciting light is collected and analyzed through a camera, the phase area ratio of Fresnel lenses with different focal lengths loaded on the spatial light modulator is changed, and the axial intensity modulation of the exciting light is realized.
Wherein the steps are as follows: the imaging result is collected through the camera, the light intensity change of the laser sheet in the sample transmission process is analyzed, the phase area ratio of Fresnel lenses with different focal lengths loaded on the spatial light modulator is changed, the axial intensity modulation of the excitation light is realized, and the problem of light intensity attenuation caused by absorption in the deep part of the sample is compensated.
In practical application, a sample imaging result is used for carrying out data analysis to calculate the light intensity change of an excitation light sheet in a transmission process, an exponential attenuation curve of the light intensity in a biological sample medium is fitted according to the Lambert beer law, collected images need to be subjected to data statistics and analysis, firstly, the axial attenuation ratio of the light intensity of the images in different areas is found, parameters of the light intensity along the axial exponential attenuation are fitted according to the parameters, and then the size of a sector area corresponding to Fresnel lens phases with different focal lengths in a modulation period on a liquid crystal panel of a spatial light modulator is adjusted to generate intensity compensation, so that the aim of uniformly distributing the light intensity along the axial direction after the attenuation effect is superposed is fulfilled.
The light intensity index attenuation curve in the biological sample medium needs to be measured by imaging analysis of a uniformly spliced light sheet, and the uniformly spliced light sheet is generated by loading Fresnel lens phases with different focal lengths in the same area size by a spatial light modulator.
And finally, the light sheet is required to be adjusted according to the light intensity attenuation curve in the biological sample medium, and the light intensity attenuation parameters at the focuses at different axial positions, which are measured by using the uniform splicing light sheet imaging analysis, correspond to the attenuation coefficients at different positions in the current sample. The pattern loaded on the spatial light modulator needs to be adjusted, different areas on the spatial light modulator correspond to different axial focuses at the sample space, and the size of the phase area of the Fresnel lens on the spatial light modulator corresponds to the light intensity distribution at different focuses.
The final light sheet is generated by the phase of the spatial light modulator, the final phase is the phase of the fan-shaped regional Fresnel lens in a polar coordinate periodic form, the area distribution of different fan-shaped regions corresponds to the light intensity distribution at different focuses, the light sheet with preset light intensity uniformly distributed along the axial direction is used for primary imaging to obtain a light intensity attenuation image of the laser light sheet transmitted in a sample, the areas of all fan-shaped regions of the light sheet phase with the generated light intensity uniformly distributed along the axial direction are equal, and at the moment, the light intensities of different focuses are the same. The light sheet generated by the method is used for absorbing the light sheet image of the tissue to compensate the attenuation of the light intensity energy at the propagation depth.
The embodiment of the present invention is described by taking the optical path in fig. 1 as an example, and details of the embodiment of the present invention are not repeated herein in specific implementation.
In summary, the embodiment of the invention uses the spliced optical sheet with adjustable axial intensity of the excitation light to solve the problem that the attenuation of light intensity energy at a propagation depth cannot be compensated when a common optical sheet works.
Example 2
The scheme of example 1 is further described below with reference to specific device parameters, as described in detail below:
as shown in fig. 1, taking a light sheet microscopic optical path in the prior art as an example, a modulation element used in a design example of the embodiment of the present invention is a spatial light modulator of Thorlabs corporation, model number EXULUS-4K1, a region that can be modulated by a liquid crystal panel of the spatial light modulator is 14.36mm × 8.08mm, a laser light source generates a gaussian light beam, the gaussian light beam firstly passes through a half-wave plate and then enters a polarization beam splitter prism, incident light is changed into linearly polarized light in a fixed direction, and incident light intensity can be adjusted through the half-wave plate. Since the modulatable size of the liquid crystal panel is 14.36mm × 8.08mm, the embodiment of the present invention expands the light beam to 8mm × 8 mm. The light beam is expanded into a Gaussian light beam with the diameter of 8mm through a beam expanding system consisting of a lens 6 and a lens 8, a small hole is arranged at a focus between two convex lenses of the beam expanding system for spatial filtering, the purpose is to optimize the quality of the emergent light beam, and the pinhole 7 is essentially a low-pass filter arranged on the Fourier plane of the lens, so that some stray light can be filtered by the method.
The light source 1 emits laser, the whole light intensity of a subsequent light path can be changed in a combined mode through the first lambda/2 glass slide 2 and the polarizing beam splitter 3, and meanwhile light emitted by the polarizing beam splitter 3 is linearly polarized light in a fixed direction. The diaphragm 4 adjusts the diameter of the light beam at this time, and the linearly polarized light in the fixed direction is adjusted to form an angle of 45 degrees with the original polarization direction through the second lambda/2 wave plate 5, and the polarization direction of the linearly polarized light is the same as the polarization direction of the optical axis of the liquid crystal panel (required by the operation of the spatial light modulator). A pinhole 7 is placed at the focal position of the beam expanding system formed by the first lens 6 and the second lens 8 for spatial filtering. The light beam is subjected to beam shaping through the first lens 6, the pinhole 7 and the second lens 8 and enters a liquid crystal panel of the spatial light modulator 9, and a multi-axial focus Fresnel lens phase pattern is loaded on the liquid crystal panel of the spatial light modulator 9. The third lens 10 and the fourth lens 11 constitute a relay device, which relays the light beam to the galvanometer 12, and the third lens 10 and the fourth lens 11 reduce the beam to adapt to the size of the galvanometer 12. The galvanometer 12 is placed at the focal position of the fifth lens 13, adjacent lenses among the fifth lens 13, the sixth lens 14, the seventh lens 16 and the eighth lens 17 form a 4F system, and light beams pass through 4 lenses to obtain required light beams at the input plane of the illumination objective lens 18. The scanning beam is focused by the illumination objective 18 to form a scanning light sheet at the focal position of the illumination objective 18 to illuminate the sample. Fluorescence signals generated by excitation form a microscopic imaging light path through the imaging objective lens 19 and the tube lens 20, and light with other wavelengths except fluorescence filtered by the optical filter 21 is collected at the camera 22.
The fifth lens 13 and the sixth lens 14 are adjacent lenses to form one system, and the seventh lens 16 and the eighth lens 17 are adjacent lenses to form another system. The 2 systems of the fifth lens 13, the sixth lens 14, the seventh lens 16 and the eighth lens 17 function as a relay lens for relaying the pattern of the galvanometer 12 to the illumination objective lens 18.
And determining the phase pattern loaded on the spatial light modulator according to the actually required light beam generation mode and the spatial light modulator spliced light beam axial offset parameter. As shown in fig. 3, in the case that the spatial light modulator is not required to implement an additional modulation function, several simple loading phases are as shown in fig. 3, the liquid crystal panel of the spatial light modulator is periodically divided into different fan-shaped areas, and the phase modulation patterns loaded in the different fan-shaped areas are distinguished by parameters corresponding to the phase of the fresnel lens.
The regional basis is that if n different axial focuses with uniform intensity need to be spliced, the sector included angle of the corresponding phase in a single period is alphasThe period of all different axial focus phases is α ═ n αsNeeds to satisfy kn alphasK α is 2 pi, k is an integer. In the process of actually designing the phase diagram, k is made as large as possible, and the corresponding alpha is made as small as possible, but the actual size of the pixel of the liquid crystal panel of the spatial light modulator needs to be considered at the same time.
The phase diagram loaded in different regions is determined according to the offset of the corresponding focus, a focal length function f (delta x) of the offset lens is constructed, and the complex amplitude distribution is according to a formula
The spatial light modulator phase modulation uses a phase portion to which a complex amplitude E is loaded. The phase of the superimposed Fresnel lens is practically equal to that of the lens optical element, and the phase of the Fresnel lens introduced with different focal lengths is equal to that of a normally propagated light beam passing throughThe convex lens converges to focus the light beam before the original focus, or the concave lens diverges to focus the light beam after the original focus. And deducing a preset focal length parameter f and an offset delta x to form an inverse proportional function relation according to a formula of the phase of the Fresnel lens and the focal length of the Fresnel lens, and verifying that the parameter f corresponds to the offset delta x through experiments.
The offset lens focal length function f (δ x) of the embodiment is measured according to experiments, namely, the offset δ x is measured by inputting different parameters, an inverse proportional function of the offset lens focal length function f and the offset δ x can be deduced by a combined lens focal length formula, and each corresponding specific parameter is obtained by using an interpolation method for the offset δ x corresponding to the measured parameter f.
Alternatively, the preset beam input to the liquid crystal panel of the spatial light modulator is not limited to a gaussian beam, and may be input such as: the invention has the advantages that the special light beams can be spliced by directly loading Fresnel lens phases with different focal lengths in different regions by the spatial light modulator, and the invention has universal applicability.
Optionally, if other light sheet forms other than the gaussian light sheet are used, the spatial light modulator may be selected to generate other light beams through phase modulation, and the functions of the spatial light modulator used in the embodiment of the present invention may be superimposed, for example: the complex amplitude of the Airy beam can be generated by selectively using the spatial light modulator to generate the Airy beam and the Bessel beam and performing axial multi-focus splicing
Multifocal phase for use with embodiments of the invention
Superposition, complex amplitude E ═ Ea×EmI.e. phase
Dividing the obtained modulation phase by 2 pi to obtain the remainder, adjusting the value to 0-1, and loading the output image onto the spatial light modulator, wherein the light beams generated by different fan-shaped regions are focused onAnd the multi-focus light beams are spliced at different axial positions.
And shaping the modulated spliced light beam by the optical system again, scanning by a galvanometer to form an optical slice, and processing the front focus of a subsequent light path lens by the galvanometer to form an f-theta galvanometer system to generate a uniform polished section. Meanwhile, in the input plane of the objective lens, the light beam vibration only changes the incidence angle, and the light sheets are still parallel up and down at the position of the biological sample.
The sample is excited for the first time by using the light sheet with preset light intensity uniformly distributed along the axial direction, a camera is used for collecting a fluorescence signal emitted by the biological sample to obtain a light intensity attenuation image of the excited light sheet propagating in the sample, and light intensity attenuation parameters of the light sheet along the propagation direction are obtained according to an imaging result. Firstly, the ratio of the light intensity images in different areas to the axial attenuation is found, the exponential attenuation curve of the light intensity in the biological sample medium is fitted according to the Lambert beer law by using the attenuation parameter, and a light intensity attenuation model for the current biological sample is constructed.
As shown in fig. 3, in the embodiment of the present invention, the compensated light intensity ratios at different axial focus positions are determined according to the attenuation parameters of the different sample models, dense sector areas on the spatial light modulator are redistributed, and the sector areas occupied by different focuses are adjusted according to the energy ratios required by the different focuses, that is, the corresponding included angles are adjusted. For example: the included angle of the fan-shaped angle of the single area corresponding to different focuses is alphaiAll the periods of different axial focal phases are alpha1+…+αnAt this time, α1Is adjusted to the light intensity of its focus by adjusting alphaiThe size of the spliced light sheet can control the axial light intensity distribution of the spliced light sheet.
FIG. 4 shows the experimental determination of fluorescent microspheres by using the embodiment of the present invention, which compares the actual fluorescent particle imaging XZ direction results of the application of the general uniform spliced optical sheet and the intensity compensation optical sheet generated by the embodiment of the present invention. The imaging is set to 10 times of exciting objective lens and 10 times of detecting objective lens, and the step length of optical sheet scanning is 0.5 micron. According to the fluorescent microsphere imaging result with or without intensity compensation, the intensities of the two types of fluorescent microspheres imaged by the optical sheets are basically consistent when the optical sheets are transmitted to the shallow layer, and the imaging of the uniform optical sheets is obviously reduced compared with a compensating optical sheet due to the attenuation of the intensity of the optical sheets when the optical sheets are transmitted to the deep layer. The experimental determination of the fluorescent microspheres proves that the splicing method with the adjustable axial intensity of the exciting light can compensate the problem of the attenuation of the light intensity energy at the deep propagation position, improve the signal background ratio of the deep tissue imaging and optimize the imaging quality.
The invention belongs to the technical field of microscopic imaging, and discloses an exciting light axial intensity adjustable splicing method based on optical sheet imaging. The different imaging requirements of different attenuation biological samples can be met aiming at different biological tissue samples. The embodiment of the invention redistributes the input light intensity along the axial direction, which is different from the method that the absorption of a medium is compensated by simply increasing the power of exciting light, and the additional light damage of the tissue shallow layer can not be caused. The splicing method is characterized in that a circular area modulated by a spatial light modulator is subdivided into dense sector areas, and the period of phase change is determined according to the number of required focuses. Meanwhile, the ratio of the axial attenuation of the light intensity of the image in different areas is found according to the imaging result of the primary sample, the parameter of the light intensity along the axial exponential attenuation is fitted according to the parameter, and then the size of the sector area corresponding to the phase of the Fresnel lens with different focal lengths in one modulation period on the liquid crystal panel of the spatial light modulator is adjusted to generate intensity compensation, so that the aim of uniformly distributing the light intensity along the axial direction again after the attenuation effect is superposed is fulfilled. The invention provides a light intensity attenuation compensation method, which can redistribute the axial intensity of a light sheet and solve the problem that the light intensity energy attenuation of a high absorption medium transmission depth can not be compensated when a common light sheet works. Further application in the field of light sheet microscopic imaging is realized.
In the embodiment of the present invention, except for the specific description of the model of each device, the model of other devices is not limited, as long as the device can perform the above functions.
Those skilled in the art will appreciate that the drawings are only schematic illustrations of preferred embodiments, and the above-described embodiments of the present invention are merely provided for description and do not represent the merits of the embodiments.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. An excitation light axial intensity adjustable splicing method based on optical sheet imaging is characterized by comprising the following steps:
shaping and filtering light beams emitted by a light source into light beams with preset parameters, and loading Fresnel lens phases with different focal lengths by using the light beams to perform phase modulation;
the light beam forms a light sheet through the scanning galvanometer and is used for exciting the sample to generate a fluorescence signal;
the light intensity change in the process of transmitting the exciting light is collected and analyzed through the camera, the phase area ratio of Fresnel lenses with different focal lengths loaded on the spatial light modulator is changed, the axial intensity modulation of the exciting light is realized, and the problem of light intensity attenuation caused by absorption in the depth of a sample is compensated.
2. The splicing method for the axial intensity of excitation light based on light sheet imaging as claimed in claim 1, wherein the light beam with preset parameters is generated before entering the spatial light modulator or is generated by modulation on the spatial light modulator,
the spatial light modulator simultaneously completes the tasks of generating a preset light beam and loading the phase of the Fresnel lens in different areas through phase modulation, finally the spatial light modulator generates spliced complex light beams with adjustable axial intensity, and the spliced complex light beams are scanned by a galvanometer to form light sheets.
3. The method for splicing excitation light with adjustable axial intensity based on light sheet imaging according to claim 1, wherein the method further comprises: the circular area modulated by the spatial light modulator is subdivided into dense sector areas, and the period of phase change is determined according to the number of focuses.
4. The splicing method with adjustable axial intensity of excitation light based on light sheet imaging as claimed in claim 1,
the light beam is generated by the phase of the spatial light modulator, and the final phase is a sector area Fresnel lens phase in a polar coordinate periodic form;
and the area distribution of different fan-shaped areas corresponds to the light intensity distribution at different focuses, and a light intensity attenuation image of the laser sheet transmitted in the sample is obtained by primary imaging of the light sheet with preset light intensity uniformly distributed along the axial direction.
5. The splicing method for the axial intensity of the excitation light based on the optical sheet imaging as claimed in claim 1, wherein the light beams with the preset parameters are: the spatial light modulator comprises a Gaussian beam, a Bessel beam, an Airy beam and a rotating Airy beam, wherein the beams generated by different fan-shaped areas of the spatial light modulator are focused at different axial positions, and Fresnel lens phases with different focal lengths are loaded in different areas, so that the splicing of different beams is realized.
6. The light sheet imaging-based excitation light axial intensity adjustable splicing method according to claim 1, wherein the method obtains light intensity attenuation parameters of a light sheet along a propagation direction according to an imaging result, finds out light intensity images in different areas along an axial attenuation ratio, fits an exponential attenuation curve of light intensity in a biological sample medium according to a Lambert beer law, and constructs a light intensity attenuation model for a current biological sample.
7. The splicing method for the axial intensity of the excitation light based on the light sheet imaging as claimed in claim 6, wherein the method determines the compensated light intensity ratios at different axial focus positions according to the attenuation parameters of different sample models, redistributes the dense sector areas on the spatial light modulator, adjusts the sector areas occupied by different focuses according to the energy ratios required by the different focuses, and controls the effect of the axial light intensity distribution of the spliced light sheet.
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