CN114153061B - Excitation optical axial intensity adjustable splicing method based on light sheet imaging - Google Patents
Excitation optical axial intensity adjustable splicing method based on light sheet imaging Download PDFInfo
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Abstract
The invention discloses an excitation optical axial intensity adjustable splicing method based on light sheet imaging, which comprises the following steps: shaping and filtering a light beam emitted by a light source into a light beam with preset parameters, and carrying out phase modulation by using phases of Fresnel lenses with different focal lengths loaded by the light beam; the light beam forms a light sheet through scanning the galvanometer and is used for exciting a sample to generate a fluorescent signal; the light intensity change in the excitation light transmission process is collected and analyzed through a camera, the duty ratio of the Fresnel lens phase areas with different focal lengths loaded on the spatial light modulator is changed, the excitation light axial intensity modulation is realized, and the light intensity attenuation problem caused by absorption in the depth of a sample is compensated. The invention reduces the energy attenuation of the light sheet in the sample propagation process, improves the spatial resolution of imaging in the depth of the sample, 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 excitation optical axial intensity adjustable splicing method based on optical sheet imaging.
Background
The light sheet fluorescence microscopy imaging technology (Light Sheet Fluorescence Microscopy, LSFM) is a new technology which combines resolution and imaging depth and is closest to the optimal target for optimizing the performance of an optical microscope, and the technology utilizes a laser light sheet with a certain thickness to excite contrast agent inside a sample to emit light, and then collects fluorescence signals in a direction perpendicular to the plane of the light sheet. Owing to the advantages, 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 a plurality of life science research fields such as brain science, organoids and the like.
In the past decade, the technology of optical sheet illumination imaging has been rapidly developed, and is continuously combined with other advanced optical imaging concepts. In the field of optical sheet imaging, a thin optical sheet is required to ensure the axial resolution of imaging, and a long optical sheet is required to ensure the field of view of imaging, but the two are contradictory. 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 trajectory of the gaussian beam is hyperbolic, and the rayleigh length is very short, which means that the area where the thickness is kept relatively thin is very limited. Thereafter, as scanning galvanometers are introduced into the field of light sheet imaging, more and more light sheet microscopy systems scan an incident light beam through an f-theta lens to generate an excitation light sheet, which can more easily combine non-diffracted light beams, such as Bessel beams, airy beams, with light sheet imaging, and at the same time, utilize dynamic scanning to generate a pattern of light sheets to make illumination more uniform.
Although the use of a scanned, non-diffracted beam can optimize the axial resolution and field of view of the light sheet, the contradiction between the two persists. Along with the continuous optimization of an excitation light path, an adjustable lens system is additionally arranged at an excitation end to move the focus position of a Gaussian beam along the beam propagation direction, so that the spliced light sheet is a feasible technical means. Currently, methods for generating an axially spliced optical sheet include axial scanning beam focus generation using a variable lens system, multilayer beam splitter axial focus position change generation, spatial light modulator phase modulation to axially focus a beam at different positions, and the like.
Although the field of view is effectively expanded by using the methods, in the actual biological tissue imaging process, most of samples are high-absorption media composed of water, fat and the like, and have strong absorption effect on light, so that the light sheet has serious energy attenuation in the sample propagation process, and finally the problems of low spatial resolution, high background noise, poor contrast and the like of the deep imaging of the samples are caused, so that the current light sheet imaging field still faces the problem that the light intensity attenuation of excitation light in the absorption media is not compensated.
On the other hand, simply relying on increasing the excitation light power to compensate for the absorption of the medium causes additional photodamage to the shallow tissue and cannot redistribute the light intensity.
Disclosure of Invention
The invention provides an excitation optical axial intensity adjustable splicing method based on optical sheet imaging, which solves the problem that the light intensity attenuation of excitation light in an absorption medium is not compensated in the existing optical sheet imaging field, reduces the energy attenuation of the optical sheet in the sample propagation process, improves the spatial resolution of imaging in the depth of a sample, reduces the image background noise, improves the contrast ratio, and is described in detail below:
an excitation optical axial intensity adjustable splicing method based on optical sheet imaging, the method comprising:
shaping and filtering a light beam emitted by a light source into a light beam with preset parameters, and carrying out phase modulation by using phases of Fresnel lenses with different focal lengths loaded by the light beam;
the light beam forms a light sheet through scanning the galvanometer and is used for exciting a sample to generate a fluorescent signal;
the light intensity change in the excitation light transmission process is collected and analyzed through a camera, the duty ratio of the Fresnel lens phase areas with different focal lengths loaded on the spatial light modulator is changed, the excitation light axial intensity modulation is realized, and the light intensity attenuation problem 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 modulated and generated 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 a zoned mode through phase modulation, and finally generates a spliced complex light beam with adjustable axial intensity through the spatial light modulator, and the spliced complex light beam is scanned through a galvanometer to form a light sheet.
Further, the method further comprises: the circular area modulated by the spatial light modulator is subdivided into dense sector areas, and the period of the phase change is determined according to the number of focuses.
In one embodiment, the beam is generated by a spatial light modulator phase, the final phase being a segmented Fresnel lens phase in the form of a polar period;
the area distribution of different sector areas corresponds to the light intensity distribution of different focuses, and a preset light intensity is used for primary imaging of a light sheet with the light intensity uniformly distributed along the axial direction to obtain a light intensity attenuation image of the excitation light sheet transmitted in a sample.
Wherein, the light beam of the preset parameters is: gaussian beams, bessel beams, airy beams and rotating Airy beams, wherein the beams generated in different fan-shaped areas of the spatial light modulator are focused at different axial positions, and different Fresnel lens phases with different focal lengths are loaded in different areas to realize the splicing of different beams.
In one embodiment, the method obtains light intensity attenuation parameters of a light sheet along the propagation direction according to an imaging result, finds the axial attenuation ratio of light intensity images of different areas, fits an exponential attenuation curve of light intensity in a biological sample medium according to lambert law, and constructs a light intensity attenuation model for a current biological sample.
Further, the method determines the compensated light intensity ratio at different axial focus positions according to 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 ratio required by different focuses, and can control the effect of 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, airy light beams and the like can be spliced axially besides the traditional Gaussian light beams;
2) The method provided by the invention can be used for matching the attenuation influence of different biological tissue samples on the light sheet, and can meet different imaging requirements of different biological samples; on the other hand, unlike simply relying on increasing the excitation light power to compensate for the absorption of the medium, the invention actually redistributes the input light intensity along the axial direction without causing additional photodamage to the shallow tissue;
3) The method provided by the invention is simple to operate, and the pattern loaded on the liquid crystal panel of the spatial light modulator can be adjusted according to the attenuation information of different axial positions in the sample obtained by uniformly splicing the light sheets;
4) The phase patterns finally loaded on the spatial light modulator are periodically arranged in sector areas with the corresponding number of focuses, so that the input original light beams are uniformly distributed and modulated to generate axial spliced light beams;
5) The invention does not conflict with other functions of the spatial light modulator, and only needs to superimpose the phases of the Fresnel lenses with different focal lengths in different areas on the existing phase modulation area, for example, the operations of generating modulated light beams, superimposing blazed gratings and the like by using the spatial light modulator can still be performed, and the invention does not conflict with the excitation optical axial intensity adjustable splicing method of the optical sheet imaging.
The axial strength adjustable splicing method 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 splicing adjustable intensity of an excitation optical axis based on optical sheet imaging;
FIG. 2 is a flow chart of the excitation optical axial intensity adjustable splicing method based on the optical sheet imaging;
FIG. 3 is a schematic diagram of loading phase and the generated scanning light slice xz direction in the splicing method provided by the invention;
FIG. 4 is a schematic diagram of the result of the intensity compensation light sheet fluorescent particle imaging XZ direction generated by the splicing method provided by the invention.
In the drawings, the list of components represented by the various numbers is as follows:
1: a light source; 2: a first lambda/2 slide;
3: a polarizing beamsplitter; 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: vibrating mirror;
13: a fifth lens; 14: a sixth lens;
15: a reflective mirror; 16: a seventh lens;
17: an eighth lens; 18: an illumination objective;
19: an imaging objective; 20: a cylindrical mirror;
21: a light filter; 22: and 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 will be described in further detail below.
Aiming at the problem that the optical sheet imaging technology is in the actual biological tissue imaging process, namely that a biological sample is mostly a high-absorption medium composed of water, fat and the like, the biological sample has a strong absorption effect on light, so that the energy of the optical sheet is seriously attenuated in the sample propagation process.
The embodiment of the invention provides an excitation optical axial intensity adjustable splicing method based on light sheet imaging, which can fully utilize a current formed light path, basically does not change the original light path setting, only needs to add a spatial light modulator in the light path to carry out phase modulation on an input light beam, and can generate various types of light beams by using an optical element without limiting Gaussian light beams.
Example 1
An excitation optical axial intensity adjustable splicing method based on optical sheet imaging, see fig. 1-4, comprises the following steps:
101: shaping and filtering the light beam emitted by the light source into a light beam with preset parameters;
the laser beam-expanding system, the spatial filtering system and other shaping devices can be used for generating a path of beam with preset parameters.
In practical application, the light beam with preset parameters can be generated before entering the spatial light modulator, and can also be modulated on the spatial light modulator, at the moment, the spatial light modulator can simultaneously complete the tasks of generating the preset light beam and loading the Fresnel lens phase in the sub-areas through phase modulation, and finally, the spatial light modulator can completely generate the spliced complex light beam with adjustable axial intensity, and the spliced complex light beam is scanned through the galvanometer to form the light sheet. 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 phases of Fresnel lenses with different focal lengths are loaded by utilizing light beams to carry out phase modulation;
namely, the phases of the Fresnel lenses with different focal lengths in the sub-areas are loaded, and the lasers in different modulation areas are focused at different positions along the axial direction, so that the laser splicing is realized. During operation, the modulated spliced light beam is shaped by the optical system again, an optical slice is formed by scanning through the vibrating mirror, the front focus of the subsequent optical path lens is processed by the vibrating mirror to form an f-theta vibrating mirror system, a uniform light sheet is generated, and meanwhile, the incident angle of the light beam is only changed by vibration on the input plane of the objective lens.
The superimposed Fresnel lens phases are actually equivalent to lens optical elements, and the Fresnel lens phases with different focal lengths are introduced to be equivalent to normally transmitted light beams, and the light beams are focused before an original focus through converging of a convex lens or are focused after the original focus through diverging of a concave lens. According to the formula of the Fresnel lens phase and the focal length thereof, the inverse proportion function relation between the preset focal length parameter f and the offset delta x is obtained, and the offset delta x corresponding to the parameter f can be verified through experiments.
103: the light beam forms a light sheet through scanning the galvanometer and is used for exciting a sample to generate a fluorescent signal;
the spliced light beam is scanned to form a light sheet, and the light sheet is incident from one side of the sample to excite the sample to generate fluorescence.
104: the light intensity change in the excitation light propagation process is collected and analyzed through a camera, the duty ratio of the Fresnel lens phase areas with different focal lengths loaded on the spatial light modulator is changed, and the excitation light axial intensity modulation is realized.
Wherein, this step specifically is: the imaging result is collected through a camera, the light intensity change of an excitation light sheet in the sample propagation process is analyzed, the phase area proportion of the 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 light intensity attenuation problem caused by absorption in the depth of the sample is compensated.
In practical application, the sample imaging result is used for carrying out data analysis and calculation on the light intensity change of the excitation light sheet in the propagation process, an exponential decay curve of the light intensity in a biological sample medium is fitted according to the lambert law, the collected image is subjected to data statistics and analysis, the axial decay ratio of the light intensity of the image in different areas is needed to be found, the parameter of the exponential decay of the light intensity along the axial direction is fitted according to the parameter, and then the sizes of sector areas corresponding to the phases of Fresnel lenses with different focal lengths in one modulation period on a liquid crystal panel of the spatial light modulator are adjusted, so that intensity compensation is generated, and the aim of uniformly distributing the light intensity along the axial direction again after the superposition and decay effect is achieved.
The light intensity exponential decay curve in the biological sample medium is required to be measured by imaging analysis of a uniform spliced light sheet, and the uniform spliced light sheet is generated by loading phases of Fresnel lenses with different focal lengths and the same area size by a spatial light modulator.
And finally, the optical sheet needs to be adjusted according to the light intensity attenuation curve in the biological sample medium, and the light intensity attenuation parameters at the focal points at different axial positions measured by using the imaging analysis of the uniformly spliced optical sheet 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 Fresnel lens phase area on the spatial light modulator corresponds to the light intensity distribution amount at the different focuses.
The final light sheet is generated by a spatial light modulator phase, the final phase is a sector-shaped zoned Fresnel lens phase in a polar coordinate periodic form, the area distribution of different sector-shaped zones corresponds to the light intensity distribution of different focuses, a preset light sheet with the light intensity uniformly distributed along the axial direction is used for primary imaging to obtain a light intensity attenuation image of the excitation light sheet transmitted in a sample, the areas of all sector-shaped zones of the light sheet phase with the light intensity uniformly distributed along the axial direction are generated, and at the moment, the light intensities of the different focuses are the same. The light sheet generated by the method is used for light sheet imaging of absorption tissues to compensate light intensity energy attenuation in deep propagation.
The embodiment of the present invention is only illustrated by taking the optical path in fig. 1 as an example, and in a specific implementation, the embodiment of the present invention will not be described in detail.
In summary, the embodiment of the invention solves the problem that the light intensity energy attenuation in the deep propagation can not be compensated when the common light sheet works by using the spliced light sheet with the adjustable excitation optical axial intensity.
Example 2
The scheme of example 1 is further described below in conjunction with specific device parameters, as described in detail below:
as shown in FIG. 1, taking a light sheet microscopic light path in the prior art as an example, a modulating element used in the design example of the embodiment of the invention is a spatial light modulator of the company Thorlabs, the model EXULUS-4K1, the area of a liquid crystal panel of the spatial light modulator which can be modulated is 14.36mm×8.08mm, a laser light source generates a Gaussian beam, the Gaussian beam firstly passes through a half-wave plate and then enters a polarization beam splitter prism, the incident light is changed into linear polarized light in a fixed direction, and meanwhile, the incident light intensity can be adjusted through the half-wave plate. Since the liquid crystal panel can modulate the size to 14.36mm×8.08mm, the embodiments of the present invention expand the beam to 8mm×8mm. The beam is expanded into Gaussian beams with the diameter of 8mm by a beam expanding system formed by the lens 6 and the lens 8, a small hole is arranged at the focus between two convex lenses of the beam expanding system for spatial filtering, the aim is to optimize the quality of emergent beams, and the pinhole 7 is a low-pass filter arranged on the Fourier plane of the lens, so that stray light can be filtered out by the method.
The light source 1 emits laser, the integral light intensity of the subsequent light path can be changed through the combination of the first lambda/2 glass slide 2 and the polarization spectroscope 3, and meanwhile, the light emitted by the polarization spectroscope 3 is linearly polarized light in a fixed direction. The aperture 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, so that the linearly polarized light is the same as the polarization direction of the optical axis of the liquid crystal panel (the working requirement of the spatial light modulator). And 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 shaped by the first lens 6, the pinhole 7 and the second lens 8 and is incident on the liquid crystal panel of the spatial light modulator 9, and the liquid crystal panel of the spatial light modulator 9 is loaded with a multiaxial focal point Fresnel lens phase pattern. The third lens 10 and the fourth lens 11 form a relay device for relaying the light beam to the galvanometer 12, and the third lens 10 and the fourth lens 11 shrink the light beam to adapt to the size of the galvanometer 12. The galvanometer 12 is placed at the focal position of the fifth lens 13, and 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 the light beam passes through the 4 lenses to obtain a required light beam on the input plane of the illumination objective 18. The scanning beam is focused by the illumination objective 18, and a scanning light sheet is formed at the focal position of the illumination objective 18 to illuminate the sample. The fluorescence signal generated by excitation forms a microscopic imaging light path with a cylindrical lens 20 through an imaging objective lens 19, and light with other wavelengths except fluorescence filtered by a filter 21 is collected at a camera 22.
The fifth lens 13 and the sixth lens 14 form one system as adjacent lenses, and the seventh lens 16 and the eighth lens 17 form another system as adjacent lenses. The 2 systems of the fifth lens 13, the sixth lens 14, the seventh lens 16 and the eighth lens 17 act as relays to relay the pattern of the galvanometer 12 into the illumination objective 18.
And determining the loaded phase pattern on the spatial light modulator according to the generation mode of the actually required light beam and the axial offset parameter of the spliced light beam of the spatial light modulator. In the case that the spatial light modulator is not required to realize the additional modulation function, as shown in fig. 3, several simple loading phases are shown in fig. 3, the liquid crystal panel of the spatial light modulator is periodically divided into different sector areas, and the phase modulation patterns loaded in the different sector areas are different from each other in terms of parameters corresponding to the fresnel lens phase.
The zoning is based on the following principle that if n different axial focuses with uniform intensity are needed to be spliced, the sector included angle of the corresponding phase in a single period is alpha s The period of all different axial focal phases is α=nα s Needs to satisfy knα s =kα=2pi, k being an integer. In the actual design of the phase diagram, k is as large as possible, and the corresponding alpha is as small as possible, but the actual size of the pixels of the liquid crystal panel of the spatial light modulator needs to be considered.
The phase diagrams loaded in different areas are determined according to the offset of the corresponding focus, an offset lens focal length function f (delta x) is constructed, and complex amplitude distribution is according to a formulaThe spatial light modulator phase modulation uses a phase portion to which the complex amplitude E is to be applied. The superimposed fresnel lens phases are actually equivalent to lens optical elements, and introducing fresnel lens phases of different focal lengths is equivalent to focusing a normally propagating beam before the original focus by converging the beam through a convex lens or focusing the beam after the original focus by diverging the beam through a concave lens. According to the formula of the Fresnel lens phase and the focal length, the inverse proportion function relation between the preset focal length parameter f and the offset delta x is obtained through deduction, and the offset delta x corresponding to the parameter f can be verified through experiments.
The offset lens focal length function f (δx) in this example is measured according to experiments, that is, the offset δx is measured by inputting different parameter experiments, the inverse proportion function of the offset lens focal length function f and the offset δx can be deduced by the combined lens focal length formula, and each specific parameter corresponding to the measured parameter f and the offset δx is obtained by using an interpolation method.
Alternatively, the preset light beam inputted to the spatial light modulator liquid crystal panel is not limited to a gaussian light beam, and may be inputted such as: the special light beam can be spliced by directly loading the phases of the Fresnel lenses with different focal lengths in different areas by the spatial light modulator.
Alternatively, if other optical sheet forms other than gaussian optical sheets are used, other optical beams may be selectively generated by the spatial light modulator through phase modulation, and the functions of the spatial light modulator used in the embodiments of the present invention may be overlapped, for example: the spatial light modulator is selected to generate Airy light beam and Bessel light beam, and axial multi-focus stitching is performed, so that complex amplitude of the generated Airy light beam can be obtainedMultifocal phase +.>Superposition, complex amplitude e=e a ×E m I.e. phase->Dividing the obtained modulation phase by 2 pi for remainder, adjusting the value to 0-1, and loading the output image onto the spatial light modulator, wherein the beams generated by different sector areas are focused at different axial positions to form a multi-focus beam by splicing.
The modulated spliced light beam is shaped by an optical system again, an optical slice is formed by scanning by a vibrating mirror, the vibrating mirror processes the front focus of a subsequent optical path lens to form an f-theta vibrating mirror system, and a uniform light sheet is generated. At the same time, in the input plane of the objective lens, the beam vibration only changes the incident angle, and the optical sheets are still parallel up and down at the biological sample position.
And (3) using a preset light sheet with light intensity uniformly distributed along the axial direction to excite the sample for the first time, using a camera to collect fluorescent signals emitted by the biological sample, obtaining a light intensity attenuation image of the excitation light sheet transmitted in the sample, and obtaining light intensity attenuation parameters of the light sheet along the transmission direction according to an imaging result. Firstly, the light intensity images of different areas are found out along the axial attenuation ratio, the attenuation parameter is used for fitting an exponential attenuation curve of the light intensity in a biological sample medium according to the lambert beer law, and a light intensity attenuation model aiming at the current biological sample is constructed.
As shown in fig. 3, according to the attenuation parameters of the different sample models, the embodiment of the invention determines the compensated light intensity duty ratio at the positions of different axial focuses, redistributes the dense sector areas on the spatial light modulator, and adjusts the sector areas occupied by the different focuses according to the energy duty ratios required by the different focuses, namely adjusts the corresponding included angles. For example: the fan-shaped included angle of the single area corresponding to different focuses is alpha i Period α=α of all different axial focal phases 1 +…+α n Alpha at this time 1 Corresponding to the intensity of the focal point thereof by adjusting alpha i The size of the light source can control the axial light intensity distribution of the spliced light sheet.
FIG. 4 is a graph showing the result of the experimental measurement of fluorescent microspheres performed by using the embodiment of the present invention, comparing the result of the application of the conventional uniformly spliced light sheet with the result of the application of the intensity compensation light sheet generated by the embodiment of the present invention in the XZ direction. The imaging is set to be 10 times excitation objective lens and 10 times detection objective lens, and the scanning step length of the optical sheet is 0.5 micrometer. According to the imaging result of the fluorescent microspheres with or without intensity compensation, the intensity of the two types of optical sheets imaging fluorescent microspheres is basically consistent in a light sheet transmission shallow layer, and in a light sheet transmission deep position, the imaging of the uniform optical sheet is obviously reduced compared with that of the compensation optical sheet due to the attenuation of the intensity of the optical sheet. The fluorescent microsphere experimental measurement shows that the excitation optical axial intensity adjustable splicing method provided by the embodiment of the invention can compensate the problem of light intensity energy attenuation in deep propagation, improve the signal background ratio of deep tissue imaging and optimize imaging quality.
The invention belongs to the technical field of microscopic imaging, and discloses an excitation optical axial intensity adjustable splicing method based on optical sheet imaging. Different imaging requirements of different attenuation biological samples can be met for different biological tissue samples. The embodiment of the invention redistributes the input light intensity along the axial direction, which is different from simply compensating the absorption of the medium by increasing the excitation light power, and does not cause extra light damage of the tissue shallow layer. According to the splicing method, a 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. And meanwhile, the axial attenuation ratio of the image light intensity of different areas is found according to the imaging result of the preliminary sample, the parameter of the axial exponential attenuation of the light intensity is fitted according to the parameter, and the sizes of the sector areas corresponding to the phases of the Fresnel lenses with different focal lengths in one modulation period on the liquid crystal panel of the spatial light modulator are further adjusted, so that intensity compensation is generated, and the purpose that the light intensity is uniformly distributed along the axial direction again after the attenuation effect is overlapped is achieved. 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 in the deep propagation of a high-absorption medium cannot be compensated when a common light sheet works. Further application in the field of light sheet microscopic imaging is realized.
The embodiment of the invention does not limit the types of other devices except the types of the devices, so long as the devices can complete the functions.
Those skilled in the art will appreciate that the drawings are schematic representations of only one preferred embodiment, and that the above-described embodiment numbers are merely for illustration purposes and do not represent advantages or disadvantages of the embodiments.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (4)
1. An excitation optical axial intensity adjustable splicing method based on optical sheet imaging, which is characterized by comprising the following steps:
shaping and filtering the light beam emitted by the light source into a light beam with preset parameters, and carrying out phase modulation on the light beam with preset parameters after lambda/2 glass slide and aperture shaping and filtering by utilizing the phases of Fresnel lenses with different focal lengths loaded by the light beam;
the light beam forms a light sheet through scanning the galvanometer and is used for exciting a sample to generate a fluorescent signal;
collecting and analyzing light intensity changes in the excitation light transmission process through a camera, changing the phase area proportion of Fresnel lenses with different focal lengths loaded on a spatial light modulator, realizing the axial intensity modulation of an excitation light axis, and compensating the light intensity attenuation problem caused by absorption in the depth of a sample;
the sample imaging result is used for carrying out data analysis and calculation on the light intensity change of the excitation light sheet in the propagation process, fitting an exponential decay curve of the light intensity in a biological sample medium according to the lambert law, carrying out data statistics and analysis on the collected image, finding out the axial decay ratio of the light intensity of the image in different areas, fitting out the parameter of the light intensity decaying along the axial direction according to the ratio, adjusting the sizes of sector areas corresponding to the phases of Fresnel lenses with different focal lengths in one modulation period on the liquid crystal panel of the spatial light modulator, generating intensity compensation, and achieving the purpose of uniformly distributing the light intensity along the axial direction again after the superposition of the decay effect;
the method comprises the steps that a light sheet is generated by phase modulation through a spatial light modulator, the final phase generated on the spatial light modulator is a sector-shaped zoned Fresnel lens phase in a polar coordinate period form, the area distribution of different sector-shaped zones corresponds to the light intensity distribution at different focuses, a preset light sheet with light intensity uniformly distributed along the axial direction is used for primary imaging to obtain a light intensity attenuation image of the excitation light sheet transmitted in a sample, the areas of all sector-shaped zones of the light sheet phase with light intensity uniformly distributed along the axial direction are equal, and the generated light sheet is used for absorbing light intensity energy attenuation in the light sheet imaging compensation transmission depth of a tissue;
subdividing a circular area modulated by a spatial light modulator into dense sector areas, and determining the period of phase change according to the number of focuses; and determining the compensated light intensity ratio at the positions of different axial focuses according to attenuation parameters of different sample models, reallocating the dense sector areas on the spatial light modulator, adjusting the sector areas occupied by the different focuses according to the energy ratio required by the different focuses, and controlling the effect of the axial light intensity distribution of the spliced light sheet.
2. The method of claim 1, wherein the light beam of the preset parameters is generated before entering the spatial light modulator or is modulated 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 a zoned mode through phase modulation, and finally generates a spliced complex light beam with adjustable axial intensity through the spatial light modulator, and the spliced complex light beam is scanned through a galvanometer to form a light sheet.
3. The method for splicing adjustable intensity of excitation optical axis based on optical sheet imaging according to claim 1, wherein the light beam with preset parameters is: gaussian beams, bessel beams, airy beams and rotating Airy beams, wherein the beams generated in different fan-shaped areas of the spatial light modulator are focused at different axial positions, and different Fresnel lens phases with different focal lengths are loaded in different areas to realize the splicing of different beams.
4. The method for splicing the adjustable intensity of the excitation optical axis based on the imaging of the optical sheet according to claim 1 is characterized in that the method obtains the light intensity attenuation parameters of the optical sheet along the propagation direction according to the imaging result, finds the light intensity images of different areas along the axial attenuation ratio, fits the exponential attenuation curve of the light intensity in the biological sample medium according to the lambert beer law, and constructs the light intensity attenuation model aiming at the current biological sample.
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