CN116559126A - Complementary Bessel light drop two-photon microscopic imaging system - Google Patents
Complementary Bessel light drop two-photon microscopic imaging system Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 62
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- 230000005284 excitation Effects 0.000 claims abstract description 20
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- 230000010287 polarization Effects 0.000 claims description 20
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- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 239000004973 liquid crystal related substance Substances 0.000 description 2
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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
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G02—OPTICS
- 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
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- 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
- G02B21/0036—Scanning details, e.g. scanning stages
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6478—Special lenses
Abstract
The invention discloses a complementary Bessel optical drop two-photon microscopic imaging system, which comprises: the device comprises an excitation light source module, a Bessel light drop modulation module, a two-photon microscopic imaging module, a spatial light modulator control module and a signal acquisition/imaging and synchronous control module; the complementary Bessel optical drop two-photon microscopic imaging system provided by the invention generates two coaxial Bessel light beams with different NA through the first spatial light modulator and the second spatial light modulator in the Bessel optical drop modulation module, and simultaneously introduces different phase differences, so that the phase differencesIs interfered by coaxial double Bessel beams to form Bessel light drops and phase differenceIs a coaxial double Bessel beam interference typeThe generated Bessel light drops shift on the axial light field distribution to obtain complementary Bessel light drops, and the complementary Bessel light drops are used for exciting a two-photon fluorescence microscopic imaging system to realize quick volume imaging of biological tissues with high contrast and high spatial resolution.
Description
Technical Field
The invention belongs to the technical field of two-photon microscopic imaging, and particularly relates to a complementary Bessel light drop two-photon microscopic imaging system.
Background
The traditional two-photon fluorescence microscopic imaging system uses Gaussian beam excitation, when volume imaging is carried out, z-axis scanning is needed to be carried out, so that the imaging speed is low, but Bessel beams have unique diffraction-free characteristics and self-healing characteristics, wherein the diffraction-free characteristics are that the transverse light field distribution of the Bessel beams is kept in a longer propagation distance, and the self-healing characteristics are that the original transverse light field distribution of the Bessel beams can be recovered after a certain distance is passed after the Bessel beams encounter an obstacle in the propagation process. Therefore, the two-photon fluorescence microscopic imaging based on Bessel beam excitation has the characteristics of long depth of field and small influence of scattering, so that the rapid volume imaging can be realized, the imaging throughput of tens of times of the traditional Gaussian beam excitation two-photon microscopic imaging can be achieved for sparse marked biological tissues, and the volume imaging at the two-dimensional frame rate is realized.
However, since there are a series of concentric rings, i.e., side lobes, around the center spot of the bessel beam and the energy ratio is high, a strong background is created, which reduces the contrast of the image. The current methods for inhibiting Bessel beam sidelobes mainly comprise confocal line detection, high-order nonlinear effect, an image difference method, a light drop method and the like. The Bessel light drop is characterized in that two coaxial Bessel light beams with different NA are utilized to interfere to form standing waves, so that the effect of inhibiting side lobes is achieved, and the problem of low contrast of two-photon fluorescence microscopic imaging excited by the Bessel light beams is solved. However, bessel light drops can generate the phenomenon of alternating light and shade of axial light field distribution while inhibiting side lobe energy, and are difficult to be used in a two-photon fluorescence microscopic imaging system.
Disclosure of Invention
The invention provides a complementary Bessel light drop two-photon microscopic imaging system, and aims to solve the problem that the existing two-photon fluorescence microscopic imaging system cannot solve the problem that the Bessel light drop technology is adopted to inhibit side lobe energy and simultaneously generate axial light field distribution light and shade alternation, so that the observation result of the two-photon fluorescence microscopic imaging system is affected.
The invention is realized in such a way that a complementary Bessel light drop two-photon microscopic imaging system is applied to microscopic imaging of a sample to be detected, and comprises:
an excitation light source module; the excitation light source module generates a horizontal polarized light beam and a vertical polarized light beam by utilizing polarization beam splitting;
a Bessel light drop modulation module for modulating the horizontal polarized light beam into a light beam with a phase differenceA first Bessel droplet formed by the interference of the coaxial double Bessel beams; simultaneously modulating said vertically polarized light beam by phase difference +.>A second Bessel droplet formed by the interference of the coaxial double Bessel beams; the first Bessel light drop and the second Bessel light drop are combined to form complementary Bessel light drops, fourier spectrums of the first Bessel light drop and the second Bessel light drop are generated, and stray light of the first Bessel light drop and the second Bessel light drop is filtered;
the two-photon microscopic imaging module is used for focusing the complementary Bessel light drops to a sample to be detected through the two-photon microscopic imaging module to realize point scanning, and the two-photon microscopic imaging module is used for collecting a backward scattering fluorescent signal generated by the sample after two-photon excitation and converting the backward scattering fluorescent signal into a voltage signal;
the spatial light modulator control module is electrically connected with the Bessel light drop modulation module, and calculates and loads a phase pattern required by the Bessel light drop modulation module;
and the signal acquisition/imaging and synchronous control module is used for synchronously controlling the output and the acquisition of the fluorescence signal and reconstructing the two-photon intensity map of the obtained voltage signal.
Preferably, the bessel droplet modulation module includes:
a first spatial light modulator and a second spatial light modulator; the horizontally polarized light passes through a first spatial light modulator to generate a first Bessel light drop, and the vertically polarized light passes through a second spatial light modulator to generate a second Bessel light drop;
the second polarization beam splitter is used for combining the first Bessel light drops and the second Bessel light drops with different polarization states to obtain Bessel light beams with uniform axial light field distribution and suppressed side lobes, namely complementary Bessel light drops;
the first Fourier transform lens and the first mask plate are arranged on the back focal plane of the first Fourier transform lens; the complementary Bessel beams pass through a first Fourier transform lens to obtain Fourier spectrum and are filtered through a first mask plate;
a second optical 4f system that passes the filtered fourier spectrum to a two-photon microscopy imaging module.
Preferably, the two-photon microscopic imaging module comprises: the system comprises an XY point scanning device, a scanning lens, a sleeve lens, a microscope objective, a dichroic mirror, an optical filter, a focusing lens and a photomultiplier, wherein a sample arranged below the XY point scanning device excites fluorescent signals through a filtered Fourier spectrum and forms reverse reflection fluorescent signals, the optical filter and the focusing lens filter and focus the reflected fluorescent signals, and the photomultiplier converts the fluorescent signals received by a photosurface into voltage signals and transmits the voltage signals to a signal acquisition/imaging and synchronous control module.
Preferably, the spatial light modulator control module includes: and the controller is used for calculating and loading a phase pattern required on a spatial light modulator in the Bessel optical drop modulation module, and is electrically connected with the Bessel optical drop modulation module.
Preferably, the horizontal polarized light passes through a first spatial light modulator to generate a first Bessel light drop, the phase pattern loaded by the first spatial light modulator is obtained by the spatial light modulator control module through calculation to obtain two concentric rings and then through inverse Fourier transform, so as to generate two Bessel light beams with different NA, and after interference, standing waves with side lobes suppressed are formed, namely, the first Bessel light drop, and the axial light field distribution of the first Bessel light drop shows light and shade alternation.
Preferably, the spatial light modulator control module selects the corresponding ratio of the inner diameter and the outer diameter when the sidelobe suppression effect is optimal, and introduces a phase difference between two concentric ringsSubjecting to inverse Fourier transform to obtain phase difference +.>Is loaded onto the first spatial light modulator to generate first Bessel drops.
Preferably, the vertically polarized light passes through a second spatial light modulator to generate a second Bessel light drop, the phase pattern loaded by the second spatial light modulator is obtained by the spatial light modulator control module through calculation to obtain two concentric rings and then through inverse Fourier transform, so as to generate two Bessel light beams with different NA, and after interference, standing waves with side lobes suppressed are formed, namely, the second Bessel light drop, and the axial light field distribution of the second Bessel light drop shows light and shade alternation;
preferably, the spatial light modulator control module selects a phase difference introduced between the same two concentric ringsSubjecting to inverse Fourier transform to obtain phase difference +.>Is loaded onto a second spatial light modulator to generate second Bessel drops. .
Preferably, the excitation light source module includes: the ultrafast laser is used for generating ultrafast laser; a first optical 4f system for collimation and beam expansion; the first half wave plate is used for adjusting the linear polarization direction of laser; and the first polarization beam splitter is used for splitting the laser into a horizontal polarization state beam and a vertical polarization state beam. .
Preferably, the XY point scanning device 11 is a dual galvanometer group or a resonant galvanometer matched with a galvanometer.
Compared with the prior art, the embodiment of the application has the following main beneficial effects:
the complementary Bessel optical drop two-photon microscopic imaging system provided by the invention generates two coaxial Bessel light beams with different NA through the first spatial light modulator and the second spatial light modulator in the Bessel optical drop modulation module, and simultaneously introduces different phase differences, so that the phase differences are causedIs interfered by coaxial double Bessel beams to form Bessel light drops and phase difference +.>The coaxial double Bessel light beams of the (2) generate offset on the axial light field distribution so as to obtain complementary Bessel light drops, and the complementary Bessel light drops are used for exciting a two-photon fluorescence microscopic imaging system to realize quick volume imaging of biological tissues with high contrast and high spatial resolution.
Drawings
Fig. 1 is a control flow chart of a complementary bessel droplet two-photon microscopic imaging system provided by the invention.
Fig. 2 is a schematic structural diagram of a complementary bessel droplet two-photon microscopic imaging system provided by the invention.
Reference numerals illustrate:
101. an excitation light source module; 1. an ultrafast laser; 2. a first optical 4f system; 3. a first half-wave plate; 4. a first polarizing beam splitter;
102. a Bessel droplet modulation module; 5. a first spatial light modulator; 6. a second spatial light modulator; 7. a second polarizing beam splitter; 8. a first fourier transform lens; 9. a first mask plate; 10. a second optical 4f system;
103. a two-photon microscopic imaging module; 11. an XY point scanning device; 12. a scanning lens; 13. a sleeve lens; 14. a microobjective; 16. a dichroic mirror; 17. a light filter; 18. a focusing lens; 19. a photomultiplier tube;
104. a spatial light modulator control module; 20. a controller;
105. a signal acquisition/imaging and synchronization control module; 21. a multifunctional acquisition card and a processor;
15. and (3) a sample.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the applications herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having" and any variations thereof in the description and claims of the present application and in the description of the figures above are intended to cover non-exclusive inclusions. The terms first, second and the like in the description and in the claims or in the above-described figures, are used for distinguishing between different objects and not necessarily for describing a sequential or chronological order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The embodiment of the invention provides a complementary Bessel optical drop two-photon microscopic imaging system, as shown in fig. 1-2, which comprises:
an excitation light source module 101; the excitation light source module 101 generates a horizontal polarized light beam and a vertical polarized light beam by polarization beam splitting;
a bessel droplet modulation module 102; the Bessel drop modulation module 102 modulates the horizontally polarized light beam to a phase differenceIs a coaxial double Bessel beamA first Bessel droplet formed by interference; simultaneously modulating said vertically polarized light beam by phase difference +.>A second Bessel droplet formed by the interference of the coaxial double Bessel beams; generating Bessel light drops with complementary axial light field distribution by combining the first Bessel light drops and the second Bessel light drops, generating Fourier spectrums of the first Bessel light drops and the second Bessel light drops, and filtering stray light of the first Bessel light drops and the second Bessel light drops;
a two-photon microscopy imaging module 103; the complementary Bessel light drops are focused to the sample 15 through the two-photon microscopic imaging module 103 to realize point scanning, and the two-photon microscopic imaging module 103 collects a back scattering fluorescent signal generated by the sample 15 after two-photon excitation and converts the back scattering fluorescent signal into a voltage signal;
a spatial light modulator control module 104; the spatial light modulator control module 104 is electrically connected with the Bessel light droplet modulation module 102, and calculates and loads a phase pattern loaded on the spatial light modulator in the Bessel light droplet modulation module 102;
a signal acquisition/imaging and synchronization control module 105; the signal acquisition/imaging and synchronous control module 105 performs synchronous control on the output and acquisition of the fluorescence signal, and performs two-photon intensity map reconstruction on the obtained voltage signal;
in this embodiment, the bessel droplet modulation module 102 includes:
a first spatial light modulator 5 and a second spatial light modulator 6; the horizontally polarized light passes through the first spatial light modulator 5 to produce a first Bessel droplet, and the vertically polarized light passes through the second spatial light modulator 6 to produce a second Bessel droplet;
the second polarization beam splitter 7 is used for combining the first Bessel light drops and the second Bessel light drops with different polarization states through the second polarization beam splitter 7 to obtain Bessel light beams with uniform axial light field distribution and suppressed side lobes, namely complementary Bessel light drops;
the first Fourier transform lens 8 and the first mask plate 9 arranged on the back focal plane of the first Fourier transform lens 8; the complementary Bessel beams pass through a first Fourier transform lens 8 to obtain Fourier spectrum and are filtered through a first mask plate 9;
a second optical 4f system 10, the second optical 4f system 10 delivering the filtered fourier spectrum to a two-photon microscopy imaging module 103;
in this embodiment, the first spatial light modulator 5 and the second spatial light modulator 6 are pure phase spatial light modulators in the prior art, and the gray phase image loaded by the spatial light modulator control module 104 applies different voltages on the liquid crystal molecules according to different gray levels, so as to change the refractive index of the liquid crystal molecules to change the optical path, thereby achieving the purpose of modulating the optical field.
The two-photon microscopic imaging module 103 includes: the sample 15 placed below the XY point scanning device 11 is subjected to filtering Fourier spectrum excitation fluorescent signals and forms reverse reflection fluorescent signals, the optical filter 17 and the focusing lens 18 are used for filtering and focusing the reflected fluorescent signals, and the photomultiplier 19 is used for converting the fluorescent signals received by the photosurface into voltage signals and transmitting the voltage signals to the signal acquisition/imaging and synchronization control module 105; the two-photon microscopic imaging module 103 adopts the prior art means;
the spatial light modulator control module 104 includes: a controller 20, configured to calculate and load a phase pattern to be loaded on a spatial light modulator in the module for generating the bessel droplet modulation 102;
the signal acquisition/imaging and synchronization control module 105 comprises a multifunctional acquisition card and a processor 21; the multifunctional acquisition card and processor 21 controls the motion control of the objective table and the motion of the objective lens 14 in the two-photon microscopic imaging module, and the multifunctional acquisition card and processor 21 collects and converts fluorescent signals of the photomultiplier tube 19 in the two-photon microscopic imaging module 103 by controlling the X and Y scanning galvanometer 11 in the two-photon microscopic imaging module 103, and synchronously controls the signal output and the signal collection; reconstructing a two-photon intensity map from the voltage signal obtained by the conversion of the photomultiplier 19; the multifunctional acquisition card and the processor 21 adopt the prior art means to acquire and output signals;
as a preferred implementation of this embodiment, the horizontally polarized light passes through the first spatial light modulator 5 to generate first bessel drops, and the phase pattern loaded by the first spatial light modulator 5 is calculated by the spatial light modulator control module 104 to be the phase differenceThe two concentric rings are obtained through inverse Fourier transform to generate two Bessel beams with different NA, and standing waves with side lobes suppressed are formed after interference, namely the first Bessel drops, and the axial light field distribution of the first Bessel drops shows light and dark alternation; the vertically polarized light passes through the second spatial light modulator 6 to generate a second Bessel light drop, and the phase pattern loaded by the second spatial light modulator 6 is calculated by the spatial light modulator control module 104 to be the phase differenceThe two concentric rings are obtained through inverse Fourier transform to generate two Bessel beams with different NA, and standing waves with side lobes suppressed are formed after interference, namely second Bessel drops, and the axial light field distribution of the second Bessel drops shows light and dark alternation;
the characteristics of a bessel beam are closely related to its fourier spectrum, i.e. the radius r of the ring, and its central spot radius can be expressed as:
the non-diffraction distance of a bessel beam can be expressed as:
the effective numerical aperture of a bessel beam can be expressed as:
wherein lambda is the laser wavelength, f 1 For the focal length of the Fourier transform lens, w is the beam radius incident on the spatial light modulator, f OB Is the effective focal length of the objective lens.
The ratio r of the characteristics of Bessel drops to the Fourier spectrum, i.e. the inner diameter to the outer diameter of concentric rings 2 /r 1 Closely related, r 2 /r 1 With a value between 0 and 1, when r 2 /r 1 When=1, it appears as a standard bessel beam, i.e., without side lobe suppression effect; ratio r of inner and outer diameters 2 /r 1 Depending on the corresponding bessel drop side lobe energy duty cycle;
the spatial light modulator control module 104 selects the corresponding ratio of the inner diameter and the outer diameter when the sidelobe suppression effect is optimal, wherein r is as follows 2 /r 1 =0.6, introducing a phase difference between two concentric ringsSubjecting it to inverse Fourier transform to obtain phase differenceIs loaded onto the first spatial light modulator 5 to generate first bessel drops; introducing a phase difference between the same two concentric rings>Subjecting to inverse Fourier transform to obtain phase difference +.>Is loaded onto the second spatial light modulator 6 to generate second bessel drops;
as a preferred implementation of the present embodiment, the excitation light source module 101 includes: an ultrafast laser 1 for generating ultrafast laser light; a first optical 4f system 2 for collimation and beam expansion; a first half-wave plate 3 for adjusting the linear polarization direction of the laser light; a first polarization beam splitter 4 for splitting the laser light into a horizontally polarized light beam and a vertically polarized light beam;
in this embodiment, the ultrafast laser 1 may be a picosecond laser or a femtosecond laser, which essentially outputs an ultrashort pulse laser with extremely high instantaneous power;
as a preferred implementation manner in this embodiment, the XY point scanning device 11 may be a dual-galvanometer group or a resonant galvanometer matched with a galvanometer; the scanning lens 12 and the sleeve lens 13 are used for expanding the laser beam to fill the entrance pupil surface of the objective lens 14 and generating a spot with a fixed size in the whole view field on the plane of the sample 15; a microobjective lens 14 for focusing the excitation beam; a dichroic mirror 16 for transmitting excitation light and reflecting the backscattered fluorescent signal; the optical filter 17 is used for further filtering out other stray light except the fluorescent signal; a focusing lens 18 for focusing the divergent fluorescent signal on a photosensitive surface of the photomultiplier tube 19; a photomultiplier 19 for converting the fluorescent signal received by the photosurface into a voltage signal;
it should be noted that, for simplicity of description, the foregoing embodiments are all illustrated as a series of acts, but it should be understood by those skilled in the art that the present invention is not limited by the order of acts, as some steps may be performed in other order or concurrently in accordance with the present invention. Further, those skilled in the art will also appreciate that the embodiments described in the specification are all preferred embodiments, and that the acts and modules referred to are not necessarily required for the present invention.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention. It will be apparent that the described embodiments are merely some, but not all, embodiments of the invention. Based on these embodiments, all other embodiments that may be obtained by one of ordinary skill in the art without inventive effort are within the scope of the invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art may still combine, add or delete features of the embodiments of the present invention or make other adjustments according to circumstances without any conflict, so as to obtain different technical solutions without substantially departing from the spirit of the present invention, which also falls within the scope of the present invention.
Claims (10)
1. A complementary bessel drop two-photon microscopy imaging system comprising:
an excitation light source module; the excitation light source module generates a horizontal polarized light beam and a vertical polarized light beam by utilizing polarization beam splitting;
a Bessel light drop modulation module for modulating the horizontal polarized light beam into a light beam with a phase differenceA first Bessel droplet formed by the interference of the coaxial double Bessel beams; simultaneously modulating said vertically polarized light beam by phase difference +.>A second Bessel droplet formed by the interference of the coaxial double Bessel beams; the first Bessel light drop and the second Bessel light drop are combined to form complementary Bessel light drops, fourier spectrums of the first Bessel light drop and the second Bessel light drop are generated, and stray light of the first Bessel light drop and the second Bessel light drop is filtered;
the two-photon microscopic imaging module is used for focusing the complementary Bessel light drops to a sample to be detected through the two-photon microscopic imaging module to realize point scanning, and the two-photon microscopic imaging module is used for collecting a backward scattering fluorescent signal generated by the sample after two-photon excitation and converting the backward scattering fluorescent signal into a voltage signal;
the spatial light modulator control module is electrically connected with the Bessel light drop modulation module, and calculates and loads a phase pattern required by the Bessel light drop modulation module;
and the signal acquisition/imaging and synchronous control module is used for synchronously controlling the output and the acquisition of the fluorescence signal and reconstructing the two-photon intensity map of the obtained voltage signal.
2. A complementary bessel droplet two-photon microscopy imaging system in accordance with claim 1, wherein said bessel droplet modulation module comprises:
a first spatial light modulator and a second spatial light modulator; the horizontally polarized light passes through a first spatial light modulator to generate a first Bessel light drop, and the vertically polarized light passes through a second spatial light modulator to generate a second Bessel light drop;
the second polarization beam splitter is used for combining the first Bessel light drops and the second Bessel light drops with different polarization states to obtain Bessel light beams with uniform axial light field distribution and suppressed side lobes, namely complementary Bessel light drops;
the first Fourier transform lens and the first mask plate are arranged on the back focal plane of the first Fourier transform lens; the complementary Bessel beams pass through a first Fourier transform lens to obtain Fourier spectrum and are filtered through a first mask plate;
a second optical 4f system that passes the filtered fourier spectrum to a two-photon microscopy imaging module.
3. A complementary bessel droplet two-photon microscopy imaging system in accordance with claim 2, wherein said two-photon microscopy imaging module comprises: the system comprises an XY point scanning device, a scanning lens, a sleeve lens, a microscope objective, a dichroic mirror, an optical filter, a focusing lens and a photomultiplier, wherein a sample arranged below the XY point scanning device excites fluorescent signals through a filtered Fourier spectrum and forms reverse reflection fluorescent signals, the optical filter and the focusing lens filter and focus the reflected fluorescent signals, and the photomultiplier converts the fluorescent signals received by a photosurface into voltage signals and transmits the voltage signals to a signal acquisition/imaging and synchronous control module.
4. A complementary bessel drop two-photon microscopy imaging system in accordance with claim 3, wherein said spatial light modulator control module comprises: and the controller is used for calculating and loading a phase pattern required on a spatial light modulator in the Bessel optical drop modulation module, and is electrically connected with the Bessel optical drop modulation module.
5. The two-photon microscopic imaging system of complementary Bessel drops according to claim 4, wherein the horizontal polarized light passes through a first spatial light modulator to generate a first Bessel drop, the phase pattern loaded by the first spatial light modulator is obtained by calculating two concentric rings through inverse Fourier transform by a spatial light modulator control module, so as to generate two Bessel beams with different NA, and a standing wave with side lobes suppressed is formed after interference, namely the first Bessel drop, and the axial light field distribution of the first Bessel drop shows light-dark alternation.
6. The two-photon microscopic imaging system of claim 5 wherein the spatial light modulator control module selects the ratio of the inner and outer diameters corresponding to the best side lobe suppressing effect to introduce a phase difference between two concentric ringsSubjecting to inverse Fourier transform to obtain phase difference +.>Is loaded onto the first spatial light modulator to generate first Bessel drops.
7. The two-photon microscopic imaging system of complementary Bessel drops according to claim 6, wherein the vertically polarized light passes through a second spatial light modulator to generate second Bessel drops, the phase pattern loaded by the second spatial light modulator is obtained by calculating two concentric rings through inverse Fourier transform by a spatial light modulator control module, so as to generate two Bessel beams with different NA, and standing waves with side lobes suppressed are formed after interference, namely the second Bessel drops, and the axial light field distribution of the second Bessel drops shows light-dark alternation.
8. The two-photon microscopic imaging system of claim 7 wherein the spatial light modulator control module selects a phase difference introduced between two identical concentric ringsSubjecting to inverse Fourier transform to obtain phase difference +.>Is loaded onto a second spatial light modulator to generate second Bessel drops.
9. A complementary bessel drop two-photon microscopy imaging system in accordance with claim 8, wherein said excitation light source module comprises: the ultrafast laser is used for generating ultrafast laser; a first optical 4f system for collimation and beam expansion; the first half wave plate is used for adjusting the linear polarization direction of laser; and the first polarization beam splitter is used for splitting the laser into a horizontal polarization state beam and a vertical polarization state beam.
10. The two-photon microscopic imaging system of claim 9 wherein the XY point scanning device 11 is a dual galvanometer group or a resonant galvanometer matched galvanometer.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116736532A (en) * | 2023-08-01 | 2023-09-12 | 中国科学院长春光学精密机械与物理研究所 | Bessel two-photon microscope illumination light path unconjugated aberration correction method and system |
| CN116736532B (en) * | 2023-08-01 | 2023-10-20 | 中国科学院长春光学精密机械与物理研究所 | Bessel two-photon microscope illumination light path unconjugated aberration correction method and system |
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