CN115372322A - Super-resolution based on two-photon nonlinear effect microscopic imaging system and imaging method - Google Patents
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Abstract
The invention discloses a super-resolution microscopic imaging system and method based on a two-photon nonlinear effect. The invention utilizes the threshold value of the two-photon nonlinear effect spatially naturally forming excited probes that exceed the diffraction limit; controlling the pulse width of the laser by using a space optical phase modulator, and changing the instantaneous peak power so as to dynamically adjust the scale of an excitation probe at the center of a light spot; the invention overcomes some common defects in the prior super-resolution imaging technology, is not limited by the selection of fluorescent markers and has universality; multiple parameters are adjustable, and flexibility is achieved; only the commonly used femtosecond pulse shaping system needs to be inserted into the excitation light circuit outside the microscope, the original excitation mode of microscope exciting light is not changed, the light path is simple, and the light path of the original microscope system is not required to be modified; the peak power of the exciting light after passing through the shaping system is greatly reduced, so that the sample cannot be photodamaged or photobleached, and the method is suitable for long-time exposure imaging and living cell imaging.
Description
Technical Field
The invention relates to a super-resolution microscopic imaging technology, in particular to a super-resolution microscopic imaging system based on a two-photon nonlinear effect and an imaging method thereof.
Background
Fluorescence microscopy plays a crucial role in our understanding of the molecular structure of biological systems and the interactions of intracellular materials. However, one of the major challenges of fluorescence microscopy is that the diffraction limit of light limits its spatial resolution. The advent of super-resolution fluorescence microscopy has broken this limitation and has enabled visualization of previously indistinguishable molecular details in biological systems. These super-resolution techniques can be divided into two categories according to the imaging strategy: (1) Light source modulation, such as stimulated emission depletion microscopy (STED) and Structured Illumination Microscopy (SIM); (2) Random scintillation or wave modulation of probe molecules, such as light activated localization technology (PALM), random optical reconstruction microscopy (STORM) and optical wave super resolution imaging (SOFI). Based on these techniques, super-resolution fluorescence microscopy has been developed and is now available for three-dimensional, multi-color and live cell imaging at nanometer-scale resolution. Nevertheless, to increase the resolution of the fluorescence microscope, i.e. to reach the nanometer-scale molecular size of the fluorescent marker, further increase of the resolution limit is required. The method currently available is a technique known as MINLUX (minimum emission flux). This is a new strategy combining structural illumination and single molecule localization, with minimal photon counting to extract precise positional information of the single molecule. MINLUX can provide 1 nanometer positioning accuracy in imaging and tracking of single molecules. It has been demonstrated in DNA-Origami structures, fixed cells and living cells, and recently extended to three dimensions.
In recent years, the resolution and imaging efficiency of super-resolution fluorescence microscopy have been gradually improved, but they are still lacking in universality, system complexity and damage to samples. The above single molecule localization techniques such as STORM, PALM, etc. randomly open a sparse subset of molecules in the field of view in each illumination step, thus requiring control of the labeling density of the fluorescent dye and complicated optical systems. The SOFI provides sub-diffraction resolution based on analysis of temporal random intensity fluctuations, so that specific fluorescent markers with good blinking properties can be used without universality. The size of the STED resolution is determined by the power of the depletion ring, too high a depletion power can cause photobleaching or other irreversible photodamage to the sample; in addition, STED requires special fluorescent dyes. The MINLUX makes a great breakthrough in resolution, but the experimental system is very complex, the technical difficulty of implementation is high, and the wide application of the MINLUX is limited.
Disclosure of Invention
Aiming at the defects of the existing super-resolution fluorescence microscopy technology, the invention provides a super-resolution microscopy imaging system based on the two-photon nonlinear effect, which can further improve the resolution of a super-resolution fluorescence microscope, has universality for different fluorescence markers, has small light damage to a sample, and can simplify the design of the system, thereby greatly promoting the development of the super-resolution fluorescence microscopy technology.
One objective of the present invention is to provide a super-resolution microscopic imaging system based on two-photon nonlinear effect.
The invention is based on the two-photon nonlinear effect the super-resolution microscopic imaging system comprises: the device comprises a femtosecond pulse excitation light source, a femtosecond pulse shaping system, a spectroscope, an objective lens, a sample piezoelectric scanning table, a signal collecting system and a computer control system; wherein, the sample is placed on a sample piezoelectric scanning platform; the sample piezoelectric scanning table is connected to a computer control system; the femtosecond pulse shaping system is connected to the computer control system; the signal collection system is connected to the computer control system; the femtosecond pulse exciting light source generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components; the femtosecond pulse shaping system converts the femtosecond pulse laser into shaping pulses with adjustable time width by performing phase modulation on light with different frequency components, so that the instantaneous peak power of the femtosecond pulse laser is adjusted to be used as exciting light; after the excitation light passes through the spectroscope, the excitation light is focused by the objective lens, the focused excitation light irradiates on a sample on the surface of the sample piezoelectric scanning table, and the sample is excited to generate two-photon absorption to generate fluorescence as signal light; the signal light is collected by the objective lens, and is collected by the signal collection system after passing through the spectroscope; by utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of instantaneous power, in a spot of focused exciting light, the two-photon absorption intensity is high in the part with stronger center, and the two-photon absorption intensity is almost incapable of absorbing two photons in the edge part, and because the two-photon absorption has a threshold value, namely a two-photon excitation threshold value, two-photon absorption can only occur if the instantaneous power is larger than the two-photon excitation threshold value, so that the two-photon absorption rate at the edge of the spot is further reduced, the two-photon absorption can only occur in the center of the spot, and the area of the spot actually excited, namely the effective excitation area is smaller than the diffraction limit spot; the computer control system performs phase modulation on light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, so as to adjust the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, instantaneous peak power is reduced, the integral brightness of light spots is unchanged at the moment, but the area of the center of the light spots capable of generating fluorescence through two-photon absorption is correspondingly changed, namely the effective excitation area is reduced; the reduction of the effective excitation area means that the scanning of the single-molecule object by using more sharp excitation light as an excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved; the femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, and a noise image can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing; the computer control system obtains a group of signal light images and noise images corresponding to the position of the excitation probe on the sample through cooperatively controlling the sample piezoelectric scanning platform and the signal collecting system; moving a sample piezoelectric scanning platform to scan the sample point by point; the computer control system analyzes the multiple signal light images and the noise images through a graphic processing algorithm, so that a super-resolution image of the sample is obtained.
Furthermore, the invention also comprises an excitation beam-shrinking collimation system, the femtosecond pulse shaping system is arranged between the femtosecond pulse shaping system and the spectroscope; the exciting light passes through the exciting light beam-shrinking collimation system, so that the area of a light spot and the divergence angle of the exciting light entering a sample are reduced.
Still include the filter, set up between sample piezoelectricity scanning platform and signal collection system, the filter will filter from the exciting light that the sample reflection returned.
The signal light focusing lens is arranged between the filter plate and the signal collecting system, and the signal light is focused after passing through the signal light focusing lens.
The type of the particles in the sample is one or more of organic molecules, organic molecule aggregates, organic nanoparticles, semiconductor nanoparticles, metal nanoparticles and nanostructures.
The femtosecond pulse shaping system mainly works on the principle that each frequency component in a wide-spectrum femtosecond pulse is subjected to phase modulation, these frequencies are recombined to become the shaping pulse required by the present invention. This system uses a femtosecond pulse spatial light modulator as a core element, which has various forms such as a reflective and transmissive liquid crystal spatial light modulator, an acoustic-optical modulator, a micro electro-mechanical system, and a deformable mirror. These elements perform the function of pulse modulation by combining with other external optical elements. Taking a femtosecond pulse shaping system using a transmission type liquid crystal spatial light modulator as an example: the femtosecond pulse shaping system sequentially comprises a first reflection type grating, a first cylindrical convex lens, a liquid crystal spatial light modulator, a second reflection type grating and a second cylindrical convex lens, and the distances between the first reflection type grating, the first cylindrical convex lens and the second cylindrical convex lens are one-time focal length f, so that a 4f pulse shaping system is formed; the femtosecond pulse laser generated by the femtosecond pulse laser source passes through the first reflective grating, and light with different frequency components is spatially separated, namely, fourier transform is performed; after passing through the first cylindrical convex lens, the light with different frequency components is collimated in space to form separated parallel light beams; the light with different frequency components is incident on different pixel units on the liquid crystal spatial light modulator correspondingly; the computer control system can independently load the phase on each pixel unit on the liquid crystal spatial light modulator so as to perform phase modulation on light with different frequency components; the modulated light with different frequency components is recombined after sequentially passing through the second cylindrical convex lens and the second reflective grating to serve as excitation light.
The invention also aims to provide a super-resolution microscopic imaging method based on the two-photon nonlinear effect.
The invention discloses a super-resolution microscopic imaging method based on a two-photon nonlinear effect, which comprises the following steps of:
1) The femtosecond pulse excitation light source generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components;
2) The femtosecond pulse shaping system converts the femtosecond pulse laser into shaping pulses with adjustable time width by performing phase modulation on light with different frequency components, so that the instantaneous peak power of the femtosecond pulse laser is adjusted to be used as exciting light;
3) After the excitation light passes through the spectroscope, the excitation light is focused by the objective lens, the focused excitation light irradiates on a sample on the surface of the sample piezoelectric scanning table, and the sample is excited to generate two-photon fluorescence as signal light;
4) The signal light is collected by the objective lens, and is collected by the signal collection system after passing through the spectroscope;
5) By utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of instantaneous power, in a spot of focused exciting light, the two-photon absorption intensity is high in the part with stronger center, and the two-photon absorption intensity is almost incapable of absorbing two photons in the edge part, and because the two-photon absorption has a threshold value, namely a two-photon excitation threshold value, two-photon absorption can only occur if the instantaneous power is larger than the two-photon excitation threshold value, so that the two-photon absorption rate at the edge of the spot is further reduced, the two-photon absorption can only occur in the center of the spot, and the area of the spot actually excited, namely the effective excitation area is smaller than the diffraction limit spot; the computer control system performs phase modulation on light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, thereby adjusting the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, instantaneous peak power is reduced, the integral brightness of a facula is unchanged at the moment, but the area of the center of the facula, which can generate fluorescence through two-photon absorption, is correspondingly changed, namely, the effective excitation area is reduced; the reduction of the effective excitation area means that the scanning of the single-molecule object by using the more sharp excitation light as the excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved;
6) The computer control system controls the sample piezoelectric scanning platform to move the sample to perform point scanning, and a signal light image at the position of the excitation probe on the sample is obtained;
7) The femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, and a noise image at the position can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing;
8) Repeating the steps 6) -7) to obtain a plurality of signal light images and corresponding noise images at the position on the sample;
9) The computer control system controls the sample piezoelectric scanning table to change the position, and repeats 6) -8), and a new group of images at other positions on the sample are obtained; obtaining a complete scanning image point by point of a sample plane by scanning the spatial position;
10 A computer control system analyzes a plurality of signal light images and corresponding noise images through a graphic processing algorithm to obtain a super-resolution image of the sample.
The invention has the advantages that:
the invention utilizes the threshold value of the two-photon nonlinear effect to naturally form an excitation probe exceeding the diffraction limit in space; the pulse width of the laser is controlled by using a spatial light phase modulator, and the instantaneous peak power is changed, so that the scale of an excitation probe at the center of a light spot can be dynamically adjusted; the invention overcomes some common defects in the existing super-resolution imaging technology, and has the advantages of universality, flexibility, simple light path and small harm to samples;
universality: the method is realized based on the nonlinear effect of two-photon absorption of particles, and is suitable for organic molecules, organic molecule aggregates, organic nanoparticles, semiconductor nanoparticles, metal nanoparticles and nanostructures, so that the method is not limited by the selection of fluorescent markers and has universality;
flexibility: the multi-parameter is adjustable, for example, the number of the fluorescence signal images and the noise images acquired at each position can be flexibly adjusted to give consideration to both resolution and efficiency, and when the signal-to-noise ratio of the system is better, the noise images can be directly acquired without being collected to process the fluorescence signal images;
the light path is simple: a commonly used femtosecond pulse shaping system (4 f system) is only required to be inserted into an exciting light path outside the microscope, the original exciting mode of the exciting light of the microscope is not changed, and the light path of the original microscope system is not required to be modified;
the harm to the sample is small: after the exciting light passes through the shaping system, the peak power is greatly reduced, so that the sample cannot be photodamaged or photobleached, and the method is suitable for long-time exposure imaging and living cell imaging.
Drawings
FIG. 1 is an optical path diagram of one embodiment of a two-photon nonlinear effect based super-resolution microscopy imaging system of the present invention;
FIG. 2 is a super-resolution microscope based on two-photon nonlinear effect according to the present invention an optical diagram of a femtosecond pulse shaping system of one embodiment of an imaging system;
fig. 3 is a schematic diagram of a two-photon nonlinear effect-based super-resolution microscopic imaging system for reducing the size of an excitation probe according to an embodiment of the present invention, wherein (a) is a schematic diagram of a light spot of an excitation light before modulation, and (b) is a schematic diagram of a light spot of an excitation light after modulation.
Detailed Description
The invention will be further elucidated by means of specific embodiments in the following with reference to the drawing.
As shown in fig. 1, the super-resolution microscopic imaging system based on the two-photon nonlinear effect of the present embodiment includes: the device comprises a femtosecond pulse excitation light source 1, a femtosecond pulse shaping system 2, an excitation beam contraction collimation system 3, a spectroscope 4, an objective lens 5, a sample piezoelectric scanning table 6, a filter 7, a signal light focusing lens 8, a signal collecting system 9 and a computer control system 10; wherein, the sample is placed on the sample piezoelectric scanning platform 6; the sample piezoelectric scanning platform 6 is connected to a computer control system 10; the femtosecond pulse shaping system 2 is connected to the computer control system 10; the signal collection system 9 is connected to the computer control system 10; the femtosecond pulse excitation light source 1 generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components; the femtosecond pulse shaping system 2 performs phase modulation on light with different frequency components to change the femtosecond pulse laser into shaping pulses with adjustable time width, so that the instantaneous peak power of the femtosecond pulse laser is adjusted to be used as exciting light, the exciting light is the femtosecond light, the pulse width of the exciting light is not limited, the pulse width can be widened or compressed by the phase modulation, and the exciting light can be modulated to the proper pulse width; exciting light passes through the exciting light beam-shrinking collimation system 3, so that the spot area and the divergence angle of the focused exciting light incident to the sample are reduced; after the excitation light passes through the spectroscope 4, the excitation light is focused by the objective lens 5, the focused excitation light irradiates on a sample on the surface of the sample piezoelectric scanning platform 6, and the sample is excited to generate two-photon absorption to generate fluorescence as signal light; the exciting light reflected from the sample is filtered by a filter 7, the signal light is focused by a signal light focusing lens 8 and collected by a signal collecting system 9 after passing through a spectroscope 4; by utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of instantaneous power, in a light spot of focused exciting light, the central part is strong, the two-photon absorption intensity is high, the edge part can hardly absorb two photons, and because the two-photon absorption has a threshold value, namely a two-photon excitation threshold value, which is determined by the properties of materials, different materials have different excitation threshold values, the two-photon excitation threshold value of a sample is obtained by continuously increasing the instantaneous power of the exciting light through phase modulation in an experiment, and the two-photon absorption can only occur if the instantaneous power is greater than the two-photon excitation threshold value, so that the two-photon absorption rate at the edge of the light spot is further reduced, the two-photon absorption can only occur at the center of the light spot, the area of the actually excited light spot, namely the effective excitation area is smaller than the diffraction limit light spot, the exciting light is pulsed light, and the instantaneous power can be ensured to be higher than the two-photon excitation threshold value of the sample under the phase modulation; the computer control system 10 performs phase modulation on the light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, thereby adjusting the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, instantaneous peak power is reduced, the integral brightness of light spots is unchanged at the moment, but the area of the center of the light spots capable of generating fluorescence through two-photon absorption is correspondingly changed, namely the effective excitation area is reduced; the reduction of the effective excitation area means that the scanning of the single-molecule object by using the more sharp excitation light as the excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved; the femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, a noise image can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing; the computer control system 10 cooperatively controls the sample piezoelectric scanning platform 6 and the signal collecting system 9 to obtain a group of signal light images and noise images corresponding to the position of the excitation probe on the sample; moving the sample piezoelectric scanning platform 6 to scan the sample point by point; the computer control system 10 analyzes the plurality of signal light images and noise images through a pattern processing algorithm to obtain a super-resolution image of the sample.
As shown in fig. 2, the femtosecond pulse shaping system 2 includes a first reflective grating 21, a first cylindrical convex lens 22, a liquid crystal spatial light modulator 23, a second reflective grating 24 and a second cylindrical convex lens 25 in sequence, and the distances therebetween are all one-time focal length f, thereby constituting a 4f pulse shaping system; the femtosecond pulse laser generated by the femtosecond pulse laser source passes through the first reflective grating 21, and the light with different frequency components is spatially separated, namely, a Fourier transform is performed; after passing through the first cylindrical convex lens 22, the light of different frequency components is collimated in space to form separate parallel beams; the light of different frequency components is incident on different pixel cells on the liquid crystal spatial light modulator 23 accordingly; the computer control system 10 is capable of independently loading phase on each pixel cell on the liquid crystal spatial light modulator to phase modulate light of different frequency components; the modulated lights with different frequency components are recombined after sequentially passing through the second cylindrical convex lens and the second reflective grating to be used as exciting lights.
The super-resolution microscopic imaging method based on the two-photon nonlinear effect comprises the following steps:
1) The femtosecond pulse excitation light source 1 generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components, in the embodiment, the center wavelength of the femtosecond pulse excitation light is 790nm, the spectral range is 740-830 nm, and the average power is about 380mW;
2) The femtosecond pulse shaping system 2 converts the femtosecond pulse laser into shaping pulses with adjustable time widths by performing phase modulation on light with different frequency components, so as to adjust the instantaneous peak power of the femtosecond pulse laser as excitation light;
3) Exciting light passes through the exciting light beam-condensing collimation system 3, so that the spot area and the divergence angle of the focused exciting light incident to the sample are reduced; after the excitation light passes through the spectroscope 4, the excitation light is focused by the objective lens 5, the focused excitation light irradiates on a sample on the surface of the sample piezoelectric scanning platform 6, and the sample is excited to generate two-photon fluorescence as signal light;
4) The exciting light reflected from the sample is filtered by a filter plate 7, the signal light is focused by a signal light focusing lens 8 and collected by a signal collecting system 9 after passing through a spectroscope 4;
5) As shown in fig. 3, by utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of the instantaneous power, in the spot of the focused excitation light, the central part is stronger, the two-photon absorption intensity is large, while the edge part can hardly absorb two photons, and because the two-photon absorption has a threshold value, i.e., a two-photon excitation threshold value, two-photon absorption can occur only if the instantaneous power is greater than the two-photon excitation threshold value, which further reduces the two-photon absorption rate at the edge of the spot, so that two-photon absorption can only occur at the center of the spot, thereby causing the actually excited spot area, i.e., the effective excitation area, to be smaller than the diffraction-limited spot, the spot size is related to the wavelength of the excitation light, which cannot be summarized, theoretically, the diffraction-limited spot radius is 0.61 λ/NA, λ is the excitation light wavelength, NA is the numerical aperture of the objective lens 5, and in this embodiment, the diffraction-limited spot radius is about 360nm; instantaneous peak power of two points A and B in the light spot of the exciting light before modulation is larger than a two-photon excitation threshold, as shown in fig. 3 (a), wherein A and B are two points in the light spot before modulation respectively, the instantaneous power is a curve changing along with time, and the instantaneous peak power represents the maximum value which can be reached by the instantaneous peak power; the computer control system 10 performs phase modulation on the light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, thereby adjusting the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, the instantaneous peak power is reduced, the integral brightness of the facula is unchanged at the moment, but the area of the center of the facula, which can generate fluorescence through two-photon absorption, is correspondingly changed, namely the effective excitation area is reduced, as shown in fig. 3 (B), the instantaneous peak power of the point A 'is reduced to be only slightly higher than the two-photon excitation threshold, the point B' is completely lower than the two-photon excitation threshold, and A 'and B' are two points in the modulated facula respectively, so the two-photon effective excitation area of the modulated facula is far smaller than the original facula; the reduction of the effective excitation area means that the scanning of the single-molecule object by using more sharp excitation light as an excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved;
6) The computer control system 10 controls the sample piezoelectric scanning platform 6 to move the sample for point scanning to obtain a signal light image at the position of the excitation probe on the sample;
7) The femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, and a noise image at the position can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing;
8) Repeating the steps 6) -7) to obtain a plurality of signal light images and corresponding noise images at the position on the sample;
9) The computer control system 10 controls the sample piezoelectric scanning table 6 to change the position, and repeats 6) -8), and a new group of images at other positions on the sample are obtained; obtaining a complete scanning image point by point of a sample plane by scanning the spatial position;
10 Computer control system 10 analyzes the multiple signal light images and corresponding noise images through a graphics processing algorithm to obtain a super-resolution image of the sample.
It is finally noted that the disclosed embodiments are intended to aid in the further understanding of the invention, but that those skilled in the art will appreciate that: various substitutions and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the embodiments disclosed, but the scope of the invention is defined by the appended claims.
Claims (6)
1. A two-photon nonlinear effect-based super-resolution microscopic imaging system is characterized by comprising: the device comprises a femtosecond pulse excitation light source, a femtosecond pulse shaping system, a spectroscope, an objective lens, a sample piezoelectric scanning table, a signal collection system and a computer control system; wherein, the sample is placed on a sample piezoelectric scanning platform; the sample piezoelectric scanning table is connected to a computer control system; the femtosecond pulse shaping system is connected to the computer control system; the signal collection system is connected to the computer control system; the femtosecond pulse exciting light source generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components; the femtosecond pulse shaping system converts the femtosecond pulse laser into shaping pulses with adjustable time width by performing phase modulation on light with different frequency components, so that the instantaneous peak power of the femtosecond pulse laser is adjusted to be used as exciting light; exciting light is focused by an objective lens after passing through a spectroscope, the focused exciting light is irradiated on a sample on the surface of a sample piezoelectric scanning table, and the sample is excited to generate two-photon absorption to generate fluorescence as signal light; the signal light is collected by the objective lens, and is collected by the signal collection system after passing through the spectroscope; by utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of instantaneous power, in a spot of focused exciting light, the two-photon absorption intensity is high in the part with stronger center, and the two-photon absorption intensity is almost incapable of absorbing two photons in the edge part, and because the two-photon absorption has a threshold value, namely a two-photon excitation threshold value, two-photon absorption can only occur if the instantaneous power is larger than the two-photon excitation threshold value, so that the two-photon absorption rate at the edge of the spot is further reduced, the two-photon absorption can only occur in the center of the spot, and the area of the spot actually excited, namely the effective excitation area is smaller than the diffraction limit spot; the computer control system performs phase modulation on light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, so as to adjust the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, instantaneous peak power is reduced, the integral brightness of light spots is unchanged at the moment, but the area of the center of the light spots capable of generating fluorescence through two-photon absorption is correspondingly changed, namely the effective excitation area is reduced; the reduction of the effective excitation area means that the scanning of the single-molecule object by using the more sharp excitation light as the excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved; the femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, and a noise image can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing; the computer control system cooperatively controls the sample piezoelectric scanning platform and the signal collecting system to obtain a group of signal light images and noise images corresponding to the position of the excitation probe on the sample; moving a sample piezoelectric scanning platform to scan the sample point by point; the computer control system analyzes the multiple signal light images and the noise images through a graphic processing algorithm, so that a super-resolution image of the sample is obtained.
2. The two-photon nonlinear effect-based super-resolution microscopy imaging system of claim 1, further comprising an excitation beam-reduction collimation system disposed between the femtosecond pulse shaping system and the beam splitter; the exciting light passes through an exciting light beam-condensing collimation system, so that the area of a light spot focused by the exciting light entering a sample and a divergence angle are reduced.
3. The two-photon nonlinear effect-based super-resolution microscopic imaging system of claim 1, further comprising a filter disposed between the sample piezoelectric scanning stage and the signal collection system, the filter filtering excitation light reflected back from the sample.
4. The two-photon nonlinear effect-based super-resolution microscopic imaging system of claim 1, further comprising a signal light focusing lens, wherein the signal light focusing lens is arranged between the filter and the signal collecting system, and the signal light is focused after passing through the signal light focusing lens.
5. The two-photon nonlinear effect-based super-resolution microscopic imaging system according to claim 1, wherein the femtosecond pulse shaping system sequentially comprises a first reflective grating, a first cylindrical convex lens, a liquid crystal spatial light modulator, a second reflective grating and a second cylindrical convex lens, and the distances between the first reflective grating, the first cylindrical convex lens, the liquid crystal spatial light modulator, the second reflective grating and the second cylindrical convex lens are all one-time focal length f, so as to form a 4f pulse shaping system; the femtosecond pulse laser generated by the femtosecond pulse laser source passes through the first reflective grating, and light with different frequency components is spatially separated, namely, fourier transform is carried out; after passing through the first cylindrical convex lens, the light with different frequency components is collimated in space to form separated parallel light beams; the light with different frequency components is incident on different pixel units on the liquid crystal spatial light modulator correspondingly; the computer control system can independently load the phase on each pixel unit on the liquid crystal spatial light modulator so as to perform phase modulation on light with different frequency components; the modulated lights with different frequency components are recombined after sequentially passing through the second cylindrical convex lens and the second reflective grating to be used as exciting lights.
6. An imaging method of the two-photon nonlinear effect based super-resolution microscopy imaging system as set forth in claim 1, wherein the imaging method comprises the steps of:
1) The femtosecond pulse exciting light source generates femtosecond pulse laser which is broadband light and comprises a plurality of different frequency components;
2) The femtosecond pulse shaping system converts the femtosecond pulse laser into shaping pulses with adjustable time width by performing phase modulation on light with different frequency components, so that the instantaneous peak power of the femtosecond pulse laser is adjusted to be used as exciting light;
3) After the excitation light passes through the spectroscope, the excitation light is focused by the objective lens, the focused excitation light irradiates on a sample on the surface of the sample piezoelectric scanning table, and the sample is excited to generate two-photon fluorescence as signal light;
4) The signal light is collected by the objective lens, and is collected by the signal collection system after passing through the spectroscope;
5) By utilizing the nonlinear characteristic that two-photon absorption is proportional to the square of instantaneous power, in a spot of focused exciting light, the two-photon absorption intensity is large in a part with a strong center, and the two-photon absorption intensity is large in a part with an edge, and because the two-photon absorption has a threshold value, namely a two-photon excitation threshold value, the two-photon absorption can only occur if the instantaneous power is larger than the two-photon excitation threshold value, so that the two-photon absorption rate at the edge of the spot is further reduced, the two-photon absorption can only occur in the center of the spot, and the area of the spot actually excited, namely the effective excitation area is smaller than the diffraction limit spot; the computer control system performs phase modulation on light with different frequency components by controlling the femtosecond pulse shaping system, and changes the time width of the femtosecond pulse laser, thereby adjusting the instantaneous peak power of the femtosecond pulse laser; the femtosecond pulse laser is widened in time, instantaneous peak power is reduced, the integral brightness of a facula is unchanged at the moment, but the area of the center of the facula, which can generate fluorescence through two-photon absorption, is correspondingly changed, namely, the effective excitation area is reduced; the reduction of the effective excitation area means that the scanning of the single-molecule object by using more sharp excitation light as an excitation probe is realized, so that the resolution of the super-resolution microscopic imaging system can be improved;
6) The computer control system controls the sample piezoelectric scanning platform to move the sample to perform point scanning, and a signal light image at the position of the excitation probe on the sample is obtained;
7) The femtosecond pulse laser is further widened in time to be completely lower than a two-photon excitation threshold value, and a noise image at the position can be obtained, so that the noise of the detector caused by the total light intensity can be kept under the condition that the total light intensity is unchanged, and the influence of the noise can be effectively eliminated in subsequent processing;
8) Repeating the steps 6) -7) to obtain a plurality of signal light images and corresponding noise images at the position on the sample;
9) The computer control system controls the sample piezoelectric scanning table to change the position, and repeats 6) -8), and a new group of images at other positions on the sample are obtained; obtaining a complete scanning image point by point of a sample plane by scanning the spatial position;
10 A computer control system analyzes a plurality of signal light images and corresponding noise images through a graphic processing algorithm to obtain a super-resolution image of the sample.
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