CN113325563B - A multicolor 3D super-resolution expansion microscope system with a large field of view - Google Patents

Abstract

The invention discloses a multi-color three-dimensional super-resolution expansion microscope system with a large field of view, belonging to the field of microscopic imaging. The system uses adaptive elements such as spatial light modulators and deformable mirrors to correct optical aberrations, while introducing dispersion The compensation component performs dispersion correction on the excitation light source. The system can avoid the influence of optical aberration on the performance of the super-resolution microscope system, and can increase the three-dimensional imaging field of view while ensuring the system resolution. Meanwhile, compared with the existing methods, the system disclosed in the present invention can easily realize multi-color imaging.

Description

A multicolor 3D super-resolution expansion microscope system with a large field of view

technical field

The invention relates to the fields of optical microscopic imaging and cell biology, and is particularly used for realizing multicolor three-dimensional super-resolution expansion microscopic imaging with a large field of view in a fluorescence microscope.

Background technique

Since the Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan W. Hell and William E. Moerner in 2014, super-resolution microscopy imaging technology has made great progress and is gradually becoming practical. In theory, the existing mainstream super-resolution microscopy imaging methods, including target switching (STED (stimulated emissiondepletion microscopy), RESOLFT), single-molecule localization (PALM, STORM, FPALM, GSDIM), saturated structured light illumination (SSIM), Fluorescence oscillation (SOFI), etc., can obtain infinitely small resolving power. When imaging biological samples, most of them can achieve a resolution level of 30-80 nm in the lateral direction and about 100 nm in the axial direction. Although the improvement of the optical system can improve the spatial and temporal resolution to a certain extent, the overall improvement effect is often limited by the fluorescence performance of the sample itself. Improvement constitutes a huge obstacle.

In 2015, Edward Boyden's laboratory published a new method called "expansion microscopy" (Expansion Microscopy, ExM), which provided a new idea for improving the resolving power of microscopic imaging technology. By physically enlarging biological samples isotropically, this method enables samples with dimensions smaller than the resolution of conventional microscopy to be resolved after the expanded state. In addition, the dilated samples are almost transparent, which greatly reduces background noise and improves the contrast of acquired images. In the following years, ExM developed rapidly, and different improved versions were proposed for different fluorescent labeling methods: including the expansion method protein-retention ExM (proExM) using traditional antibody labeling or fluorescent protein, the expansion method using in situ hybridization labeled RNA The method expansion FISH (ExFISH), the expansion method iterative ExM (iExM), which achieves extremely high resolution through repeated sample expansion, and the expansion pathology (ExPath) method for studying human tissue samples for pathological or diagnostic purposes, among others. Essentially, ExM is a sample processing method independent of specific imaging techniques and is therefore compatible with almost all existing optical microscopy imaging techniques.

In 2018, Helge Ewers laboratory combined ExM and STED super-resolution microscopy imaging technology to realize ExSTED, which further improved the resolution, achieving two-dimensional ~10nm, three-dimensional 50-70nm resolution, and saw microtubules through 2D ExSTED. tubular structure. However, if a deeper structure (a depth of 5 μm or more) is to be observed, spherical aberration occurs due to refractive index mismatch. To solve this problem, the method used by Helge Ewers' research group is to replace the oil mirror with a water mirror, which reduces the influence of spherical aberration to a certain extent, but also reduces the three-dimensional resolution from 50nm to 70nm. In addition, there are still huge challenges in this combination of technologies in practice. The main bottlenecks include: the reduction of the actual field of view caused by the expansion, the slow imaging speed and the reduction of the density of fluorescent labels, and the deterioration of performance (mainly fluorescent bleaching). and fluorescence signal reduction) problems, etc.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a multi-color three-dimensional super-resolution expansion microscope system with a large field of view, which takes into account its multi-color imaging function while ensuring that the expansion STED microscope has a large field of view.

In order to achieve the above object, the present invention provides a multi-color three-dimensional super-resolution expansion microscope system with a large field of view, the system comprising: an illumination unit and a detection unit.

The lighting unit includes a loss light path, an excitation light path and a mixed light path.

The lossy light path is sequentially provided with a lossy light source, a first light modulator, a relative phase delay component, a first reflector, a first telescopic component, a phase modulation component for phase modulation of the suppressed beam, and a second telescopic component , the second mirror, the first 1/2 wave plate, and the third telephoto component; wherein the relative phase retardation component splits the lost light beam into two mutually perpendicular polarization components and produces phase delay for these two components to destroy its coherence.

The excitation light path is sequentially provided with an excitation light source, a second light modulator, a dispersion compensation component, and a fourth telephoto component; wherein the dispersion compensation component is used to compensate the dispersion of light of different wavelengths.

The mixed light path is sequentially provided with a first dichroic mirror, a second dichroic mirror, a scanning component, a fifth telephoto component, an adaptive aberration correction component, a first 1/4 wave plate, a sixth telephoto component, Third mirror, microscope objective and sample stage. A beam splitter is arranged on the optical path in the sixth telescopic assembly, and the first dichroic mirror combines the beam of loss light from the third telescopic assembly and the beam of excitation light from the fourth telescopic assembly. into a mixed beam. The function of the scanning component is to change the deflection angle of the mixed light beam at the entrance pupil of the microscope objective lens, so that the focus of the mixed light beam realizes two-dimensional scanning on the focal plane of the microscope objective lens. The self-adaptive aberration correction component is provided with a ninth reflection mirror and a deformation mirror in sequence; after the mixed light beam is reflected by the ninth reflection mirror, it reaches the surface of the deformation mirror, and the wave front of the mixed light beam is corrected.

The detection unit includes a scattered light imaging component and a fluorescence imaging component. The mixed light beam illuminates the sample to generate a scattered signal. The scattered light imaging component is arranged on the reflected light path of the scattered signal passing through the beam splitter, and is used to detect the scattered signal of the sample. ; The fluorescence signal generated by the excitation of the mixed beam returns to the fluorescence imaging component after being corrected by the deformed mirror wavefront. The fluorescence imaging component is arranged on the transmission light path of the fluorescence signal passing through the second dichroic mirror, and is used for detecting the fluorescence signal of the sample.

Further, the phase modulation component includes a seventh reflector, a spatial light modulator, a third 1/4 wave plate, a first lens, and an eighth reflector arranged in sequence along the optical path; the first lens is located between the spatial light modulator and the eighth reflector. The middle position of the reflector, and the focal length of the first lens is equal to the distance between the first lens and the eighth reflector. The loss light first reaches the first phase map area of the spatial light modulator, and performs a phase modulation on its horizontal polarization component; then the loss light is reflected by the spatial light modulator and passes through the third 1/4 wave plate and the first lens in turn, Then it is reflected by the eighth mirror and returned, and after passing through the first lens and the third 1/4 wave plate, it reaches the second phase map area of the spatial light modulator again to perform secondary phase modulation on the vertical polarization component of the lossy light source; after modulation After the lost light is focused by the microscope objective, the light intensity in the focused spot is distributed in a hollow around the focal point. The first and second phase diagrams of the spatial light modulator are circular and are located in the horizontal center of the entire effective area of the spatial light modulator. The positions of the two phase diagrams can be exchanged, and the diameter is equal to the effective aperture of the phase modulation component.

Further, the scanning assembly includes a first scanning mirror, a second scanning mirror and a third scanning mirror arranged in sequence. The first scanning mirror has the same size as the effective aperture of the scanning assembly, and the scanning direction is perpendicular to the second scanning mirror and the third scanning mirror. The scanning frequency of the first scanning mirror is faster than that of the second scanning mirror and the third scanning mirror.

Further, the first telephoto assembly, the second telephoto assembly, the third telephoto assembly, the fourth telephoto assembly, the fifth telephoto assembly, and the sixth telephoto assembly all include two convexes that are arranged back-to-back and are confocal. convex lens. It is used to expand the beam (or reduce the beam) to collimate and maintain the conjugate relationship between the spatial light modulator, the first scanning mirror, the deformable mirror and the back focal plane of the microscope objective in the system.

Further, the present invention realizes multicolor fluorescence imaging through the light modulator, the dispersion compensation component and the fluorescence imaging component.

Specifically, the excitation light source is preferably a white light laser, and a second light modulator is placed behind it to select the wavelength of the excitation light beam and modulate the transmitted light intensity of the corresponding wavelength, and the second light modulator is preferably an acousto-optic tunable filter , the wavelength required for modulation is determined by the fluorescent dye used in the sample, and preferred wavelengths include 488 nm, 590 nm and 650 nm. The dispersion compensation component includes a third 1/2 wave plate, a third polarization beam splitter, a second 1/4 wave plate, a third dichroic mirror, a fourth dichroic mirror, and a fifth dichroic mirror, which are arranged in sequence. The relative positions of the three dichroic mirrors are determined according to the dispersion value corresponding to the excitation light wavelength, that is, determined by the emission sequence corresponding to the excitation light pulses of different wavelengths. , and finally 488nm. The three-way excitation light first passes through the third 1/2 wave plate to become vertically polarized light, then is reflected by the third polarization beam splitter, and then passes through the second 1/4 wave plate. Subsequently, the 488nm excitation light is reflected by the third dichroic mirror and returned to the original path, the 590nm excitation light is transmitted through the third dichroic mirror, and the fourth The dichroic mirror transmits and the fifth dichroic mirror reflects back the same way. After reflection, the three-way excitation light returned from the original path passes through the 1/4 wave plate again, becomes horizontally polarized light, transmits through the third polarization beam splitter, and finally is focused on the sample by the microscope objective lens to generate a fluorescent signal. Imaging components are collected. The fluorescence imaging assembly includes a sixth dichroic mirror for separating the fluorescence signal detected by the current imaging module and the fluorescence signal of the subsequent imaging module, a filter for filtering stray light signals that do not belong to the spectrum of the detection channel, and focusing the collected fluorescence to A second lens on the photon counter and a photon counter that linearly converts fluorescent signals into electrical signals according to the number of collected fluorescent photons.

Further, the present invention ensures the incoherent superposition of the two beams of loss light through the relative phase delay component, thereby realizing three-dimensional super-resolution microscopic imaging.

Specifically, the relative phase retardation component includes a second half-wave plate and a first polarization beam splitter for dividing the lost light beam into a vertical polarization component and a horizontal polarization component, and a fourth mirror for changing the direction of the vertical polarization component , a fifth mirror and a sixth mirror for changing the direction and optical path of the horizontal polarization component, and a second polarization beam splitter for combining the vertical polarization component and the horizontal polarization component.

Further, the present invention can not only be combined with the expansion microscopy technology, but also solve the aberration problem caused by the mismatch between the refractive index of the expanded biological sample and the oil immersion objective lens through an adaptive aberration correction component.

Further, the present invention can realize three-dimensional large-field microscopic imaging through the sample stage, the scanning component and the adaptive aberration correction component.

Specifically, the sample stage has the ability to move in three dimensions, and the scanning component is a three-galvo mirror system, including a first scanning mirror, a second scanning mirror and a third scanning mirror arranged in sequence along the optical path. By changing the mixed beam at the entrance pupil of the microscope objective The deflection angle of the hybrid beam can achieve two-dimensional high-speed scanning on the focal plane of the microscope objective lens, and the combination of the two can achieve three-dimensional fast scanning; the adaptive aberration correction component can solve the aberration problem caused by deep imaging, while ensuring the resolution Under the premise of the ability to achieve deep imaging, the imaging depth is increased, and the three-dimensional space large field of view microscopic imaging is realized.

Compared with the prior art, the beneficial effects of the present invention are:

(1) Simultaneous multi-color fluorescence imaging, three-dimensional super-resolution and expansion microscopy imaging capabilities;

(2) In depth imaging and dilation microscopy imaging, adaptive optics can be used to correct sample aberrations caused by refractive index mismatch;

(3) Before observing the sample, the system can correct the aberration on the optical path of the system through the spatial light modulator and the deformable mirror.

(4) Simplify the number of optical components of the existing system, the structure is simple, the system is stable, the realization difficulty is low, and the cost is low.

(5) In the present invention, by changing the deflection angle of the mixed light beam at the entrance pupil of the microscope objective lens, the focus of the mixed light beam realizes two-dimensional high-speed scanning on the focal plane of the microscope objective lens, and the combination of the two realizes three-dimensional fast scanning and high imaging speed; The adaptive aberration correction component can solve the aberration problem caused by deep imaging, and realize deep imaging on the premise of ensuring the resolution capability, thereby increasing the imaging depth, and then realizing three-dimensional space large field of view microscopic imaging.

Description of drawings

1 is a schematic structural diagram of a multicolor three-dimensional super-resolution expansion microscope system with a large field of view in an embodiment of the present invention;

2 is a schematic structural diagram of a relative phase delay component in an embodiment of the present invention;

3 is a schematic structural diagram of a dispersion compensation component in an example of the present invention;

4 is a schematic structural diagram of a phase modulation component in an embodiment of the present invention;

5 is a schematic structural diagram of a scanning component in an embodiment of the present invention;

6 is a schematic structural diagram of an adaptive aberration correction component in an example of the present invention;

7 is a schematic structural diagram of a fluorescence imaging assembly in an embodiment of the present invention;

FIG. 8 is a dispersion curve diagram of an excitation light source in an embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below with reference to the embodiments and the accompanying drawings.

Example

Referring to FIGS. 1 to 7 , the multicolor 3D super-resolution expansion microscope system with a large field of view of this embodiment includes:

Loss light source 1, excitation light source 2, first light modulator 3, second light modulator 4, relative phase delay component 5, dispersion compensation component 6, first mirror 7, first telephoto component 8, phase modulation Assembly 9, second telescopic assembly 10, second mirror 11, first 1/2 wave plate 12, third telescopic assembly 13, fourth telescopic assembly 14, first dichroic mirror 15, second dichroic Mirror 16, scanning assembly 17, fifth telescopic assembly 18, adaptive aberration correction assembly 19, first 1/4 wave plate 20, sixth telescopic assembly 21, third mirror 22, microscope objective 23, sample 24. Sample stage 25, beam splitter 26, photomultiplier tube 27, fluorescence imaging assembly 28. All optics and samples are on the coaxial optical path.

In this embodiment, the lossy light beam from the lossy light source 1 passes through the first modulator 3 and then reaches the relative phase delay component 5 . After being output from the relative phase delay component 5 , it sequentially passes through the first reflecting mirror 7 and the first telephoto component 8 , the phase modulation component 9 , the second telephoto component 10 , the first 1/2 wave plate 12 and the third telephoto component 13 reach the first dichroic mirror 15 . The excitation light beam from the excitation light source 2 passes through the second modulator 4 and then reaches the dispersion compensation component 6 , and is output from the dispersion compensation component 6 to the first dichroic mirror 15 through the fourth telephoto component 14 . The loss light beam and the excitation light beam are combined into a mixed beam by the first dichroic mirror 15 . After the mixed light beam passes through the second dichroic mirror 16 , it passes through the scanning component 17 . The mixed beam output from the scanning component 17 is re-expanded and collimated by the fifth telescopic component 18 , and the expanded and collimated mixed beam reaches the adaptive aberration correction component 19 . The mixed beam output from the adaptive aberration correction component 19 passes through the first quarter-wave plate 20 and the sixth telephoto component 21 in sequence, and is then reflected by the third mirror 22 and then enters the microscope objective lens 23, and is finally focused to the Inside the sample 24 on the sample stage 25 . The scattered signal from the sample 24 is collected in the reverse direction by the microscope objective lens 23, passes through the third mirror 22 and the beam splitter 26 in turn, and enters the photomultiplier tube 27, and is processed by the computer to form an image of the scattered sample. After the fluorescence signal from the sample 24 is collected in reverse by the microscope objective lens 23, it sequentially passes through the third mirror 22, the sixth telephoto component 21, the first quarter wave plate 20, the adaptive aberration correction component 19, and the fifth The telephoto component 18 and the scanning component 17 are finally transmitted into the fluorescence imaging component 28 by the second dichroic mirror, and are processed by the computer to form an image of the fluorescent sample.

In this embodiment, the loss light source 1 provides a loss light beam for the microscope system, and the wavelength range thereof is 770-775 nm. The average power of the loss light source 1 is greater than 3W/nm; the loss light beam is linearly polarized light with a purity higher than 10000:1.

In this embodiment, the excitation light source 2 provides an excitation light beam for the microscope system, which can be a white laser or a laser group composed of multiple solid-state lasers. This embodiment is a white laser; the wavelength range is 410-2400 nm. The average power of the excitation light source 2 is not less than 0.5mW/nm; the excitation beam is linearly polarized light with a purity higher than 1000:1.

The loss light source 1 and the excitation light source 2 may be continuous lasers or pulsed lasers. In this embodiment, the two are pulsed lasers, and the pulse frequencies of the two are between 20 and 200 MHz and keep synchronization, preferably 78 MHz. Wherein, the pulse width of the loss light source 1 should be between 500-1000ps, and the pulse width of the excitation light source 1 should be less than 100ps.

The first light modulator 3 is used to modulate the transmitted light intensity of the lost light, and the first light modulator in this example is an acousto-optic modulator. The second light modulator 4 is used to select the desired wavelength of the excitation light beam and modulate the transmitted light intensity of the corresponding wavelength. The second light modulator in this embodiment is an acousto-optic tunable filter, and the wavelength to be modulated is the one used in the sample. Determined by the fluorescent dye, preferred wavelengths include 488nm, 590nm and 650nm.

The relative phase delay element 5 is used to split the lost light beam and generate a phase delay large enough to destroy the coherence between the two components. There are various implementations of the relative phase delay component 5 . As shown in FIG. 2 , the relative phase retardation component of this embodiment includes a second half-wave plate 29 , a first polarizing beam splitter 30 , a fourth reflecting mirror 31 , a fifth reflecting mirror 32 , and a sixth reflecting mirror 33 and a second polarizing beam splitter 34 . The optical path of the loss light entering the relative phase delay element 5 is shown by the arrow in FIG. 2 , and the loss light beam first passes through the second half-wave plate 29 and reaches the first polarization beam splitter 30 . The function of the second 1/2 wave plate is to adjust the polarization direction of the loss light, so as to adjust the energy ratio of the loss light passing through the first polarization beam splitter 30 . After passing through the first polarizing beam splitter 30 , the lost light is divided into two components: horizontally polarized light and vertically polarized light, wherein the vertically polarized light component is reflected by the first polarizing beam splitter 30 and then reflected by the fourth mirror 31 . Reach the second polarizing beam splitter 34 ; the horizontally polarized light component directly passes through the first polarizing beam splitter 30 , and is then reflected by the fifth mirror 32 and the sixth mirror 33 to reach the second polarizing beam splitter 34 . The vertically polarized light component and the horizontally polarized light component are combined by the second polarization beam splitter 34 through the second polarization and then exit.

The dispersion compensation component 6 is used to perform dispersion compensation on the multiplex excitation light, so that excitation light pulses of different wavelengths are emitted simultaneously. In this embodiment, the used excitation light source 2 is a white laser, and the preferred excitation light wavelengths are 650 nm, 590 nm and 488 nm. The dispersion curve of the excitation light source 2 is shown in FIG. 8 , and it can be seen that the excitation light pulse of the long wavelength and the excitation light pulse of the shorter wavelength are preferentially emitted. Of the three preferred wavelengths, the excitation light pulse of 650 nm exits first, followed by the excitation light pulse of 590 nm, and finally 480 nm. To this end, it is necessary to perform dispersion compensation on the three excitation lights, so that the light pulses can be emitted simultaneously and reach the sample 24 at the same time. There are various implementation schemes for the dispersion compensation component 6 to realize the function. As shown in FIG. 3 , the dispersion compensation component of this embodiment includes a third 1/2 wave plate 35 , a third polarization beam splitter 36 , a second 1/4 wave plate 37 , a third dichroic mirror 38 , a fourth Chromatic mirror 39 and fifth dichroic mirror 40 . The optical path of the excitation light entering the dispersion compensation component 6 is shown in FIG. 3 , and the excitation light first passes through the third half-wave plate 35 to reach the third polarization beam splitter 36 . Among them, the function of the third 1/2 wave plate is to adjust the incident excitation light to vertically polarized light. The excitation light is reflected by the third polarization beam splitter 36 and then directly transmitted through the second 1/4 wave plate 37 . Subsequently, the 488 nm excitation light is reflected by the third dichroic mirror 38 and returned to the original path, the 590 nm excitation light is transmitted by the third dichroic mirror 38 and reflected by the fourth dichroic mirror 39 and returned to the original path, and the 650 nm excitation light is passed through the third dichroic mirror 38 The transmission, the transmission of the fourth dichroic mirror 39 and the reflection of the fifth dichroic mirror 40 return the same way. After the reflection, the three-way excitation light returned from the original path passes through the 1/4 wave plate 37 again, becomes horizontally polarized light, and is transmitted through the third polarization beam splitter 36 and then exits.

The loss light beam emitted from the relative phase retardation element 5 is reflected by the first reflecting mirror 7 and then incident on the first telescopic element 8 . The first telephoto assembly 8 includes two convex lenses that are confocal and arranged to face the back and are used for beam expansion (or beam reduction) collimation, which is effective for matching the size of the lost light beam reflected from the first mirror 7 and the phase modulation assembly 9 Aperture, whose magnification is equal to the effective aperture of the phase modulation component 9 divided by the loss light beam size after the loss light is reflected from the first reflecting mirror 7 .

The lossy light beam exiting from the first telescopic element 8 enters the phase modulation element 9 . The phase modulation component 9 is used to modulate the phase of the input loss light, and there are various implementation schemes. The phase modulation component 9 of this embodiment includes a seventh reflecting mirror 37 , a spatial light modulator 38 , a third 1/4 wave plate 39 , a first lens 40 , and an eighth reflecting mirror 41 . The first lens 40 is located at an intermediate position between the spatial light modulator 38 and the eighth reflector 41 , and the focal length of the first lens is equal to the distance between the first lens 40 and the eighth reflector 41 . The transmission direction of the loss light beam in the phase modulation component 12 is shown by the arrow in FIG. 4 , the loss light first passes through the seventh reflector 37 to reach the first phase map area of the spatial light modulator 38, and performs a first phase phase on its horizontal polarization component. Modulation; then the loss light is reflected by the spatial light modulator 38 and then passes through the third 1/4 wave plate 39 and the first lens 40 in turn, and then is reflected by the eighth mirror 41 and returns, and passes through the first lens 40 and the third 1/4 wave plate 40. After the 4-wave plate 39, it reaches the second phase map area of the spatial light modulator 38 again to perform secondary phase modulation on the vertical polarization component of the loss light source; the modulated loss light is focused by the microscope objective lens, and the light intensity in the focused spot surrounds the focus A hollow distribution.

Among them, the spatial light modulator 38 only has a phase modulation effect on linearly polarized light in a single direction, the corresponding operating wavelength range is 750-850 nm, and other visible light bands outside the corresponding operating wavelength range only exhibit high reflectivity effects, preferably Hamamatsu Corporation of Japan The X13139-02 Spatial Light Modulator. The first and second phase diagrams of the spatial light modulator are circular and are located in the horizontal center of the entire effective area of the spatial light modulator.

The loss light beam emitted from the phase modulation element 9 reaches the first dichroic mirror 15 after passing through the second telescopic element 10 , the first 1/2 wave plate 12 and the third telescopic element 13 . Wherein, the second telephoto assembly 10 and the third telephoto assembly 13 both include two confocal convex lenses disposed opposite to each other. The second telephoto assembly 10 is used for beam expansion (or beam reduction) collimation, matching the effective aperture of the phase modulation assembly 9 and the effective aperture of the scanning assembly 17, and the magnification is the effective aperture of the scanning assembly 17 divided by the phase modulation effective aperture Effective aperture of component 9. In addition, the second telescopic assembly 10 and the third telescopic assembly 13 can be used together to maintain the conjugate relationship between the phase modulation assembly 9 and the scanning assembly 17 in the system. The first 1/2 wave plate 12 is used to purify the polarization state of the loss light, so as to avoid the polarization state change of the loss light caused by the use of the mirror in the system.

The excitation light beam emitted from the dispersion delay element 6 reaches the first dichroic mirror 15 through the fourth telephoto element 14 . Wherein, the fourth telephoto assembly 14 includes two confocal confocal confocal lenses. The fourth telephoto component is used for beam expansion (or beam reduction) collimation, matching the size of the excitation light beam emitted from the dispersion delay component 6 and the effective aperture of the scanning component 17, and the magnification is the effective aperture of the scanning component 17 divided by the dispersion. The size of the excitation light beam emitted from the delay element 6 .

The loss light beam emitted from the third telescopic assembly 13 is reflected by the first dichroic mirror 15, and the excitation light beam emitted from the fourth telescopic assembly 14 directly passes through the first dichroic mirror 15, and the two beams are combined into a mixed beam . Subsequently, the mixed light beam is reflected by the second dichroic mirror 16 and enters the scanning assembly 17 .

The function of the scanning component 17 is to change the deflection angle of the mixed beam at the entrance pupil of the microscope objective 23 , so that the focus of the mixed beam scans two-dimensionally on the focal plane of the microscope objective 23 inside the sample 24 , and there are various implementation schemes. The scanning assembly 17 in this embodiment includes a first scanning mirror 42 , a second scanning mirror 43 and a third scanning mirror 44 , and the transmission direction of the mixed beam in the scanning assembly 17 is shown by the arrow in FIG. 5 . The first scanning mirror 42 is the same size as the effective aperture of the scanning assembly 17 , and the scanning direction is perpendicular to the second scanning mirror 43 and the third scanning mirror 44 . The scanning frequency of the first scanning mirror 42 is faster than that of the second scanning mirror 43 and the third scanning mirror 43 . Preferably, the scanning frequencies of the first scanning mirror 42, the second scanning mirror 43 and the third scanning mirror 44 are 16KHz, 0.1KHz and 0.1KHz, respectively.

The mixed beam from the scanning component 17 is incident on the adaptive aberration correction component 19 after passing through the fifth telephoto component 18 . Wherein, the fifth telephoto assembly 18 includes two confocal confocal confocal lenses arranged on the back. The fourth telephoto component is used for beam expansion (or beam reduction) collimation, matching the effective aperture of the scanning component 17 and the effective aperture of the adaptive aberration correction component 19 and maintaining the conjugate relationship between the two, and the magnification is the adaptive image. The effective aperture of difference correction assembly 19 is divided by the effective aperture of scanning assembly 17 .

The function of the adaptive aberration correction component 19 is to change the wavefront of the mixed beam and the fluorescence signal excited by the sample 24, so that the mixed beam can be perfectly focused on various depths of the sample 24, and the excited fluorescence can be detected by the fluorescence imaging component 28. fully received. The adaptive aberration correction component of this embodiment includes a ninth reflection mirror 45 and a deformable mirror 46 . The transmission direction of the mixed light beam in the adaptive aberration correction component 19 is shown by the arrow in FIG. 6 , the mixed light beam is reflected by the ninth reflecting mirror 45 and then reaches the deforming mirror 46 , and is reflected by the deforming mirror and then exits. The anamorphic mirror 46 has the same size as the effective aperture of the adaptive aberration correction component 19, and is preferably a DM140A-35-P01 anamorphic mirror from Boston Micromachines Corporation.

The mixed beam emitted from the adaptive aberration correction component 19 passes through the first quarter wave plate 20 and the sixth telephoto component 21, and is then reflected by the third reflecting mirror 22, then enters the entrance pupil of the microscope objective lens 23, and is finally focused into the sample 24 placed on the sample stage 25. Among them, the function of the first 1/4 wave plate is to change the polarization state of the mixed beam from linear polarization to circular polarization. The sixth telephoto assembly 21 includes two confocal convex lenses arranged on the back and confocal to expand the beam (or reduce the beam) to collimate and maintain the common rear focal plane of the adaptive aberration correction assembly 19 and the microscope objective lens 23 in the system. Yoke relationship, its magnification is equal to the size of the entrance pupil of the microscope objective divided by the effective aperture of the adaptive aberration correction component. The microscope objective 23 is used to focus the mixed beam on the sample for illumination, and collect the fluorescence from the sample in reverse; to ensure the resolution, choose a plan achromatic immersion microscope with a numerical aperture greater than 1.05 and a magnification of 60 to 100 times. objective lens. The sample stage 25 is used to carry the sample 24 and provides three-dimensional movement capability; the sample stage 25 selects a two-dimensional radial movement range greater than 5 mm, an axial movement range greater than 100 μm, and a movement accuracy of less than 1 μm.

The sample 24 can be either a scattered light sample or a fluorescent sample. For the scattered light sample, under the illumination of mixed light, the scattered light signal emitted by the sample 24 is collected by the microscope objective lens 23 in reverse. The scattered light sequentially passes through the third reflecting mirror 22 , the right lens in the sixth telephoto assembly 21 , the beam splitter 26 and the photomultiplier tube 27 . The function of the photomultiplier tube is to convert the detected scattered light signal into an electrical signal, the reddest is read by the computer, and it is restored to the scattered light sample signal. In this embodiment, the scattered light sample is gold particles, which can be used to image the loss light hollow focused spot and the excitation light solid focused spot, and use the spatial light modulator 38 to realize the shaping of the loss light focused spot and realize the combination with the excitation light Spatial alignment of a solid focused spot.

For fluorescent samples, the fluorescent signal emitted by the sample 24 is collected inversely by the microscope objective 23 under the illumination of the mixed light. After the fluorescence passes through the third reflecting mirror 22, the sixth telephoto assembly 21, the first 1/4 wave plate 20, the adaptive aberration correction assembly 19, the fifth telephoto assembly 18, and the scanning assembly 17 in sequence, it finally reaches the second and second Chromatic mirror 16 transmits into fluorescence imaging assembly 28 . Depending on whether multicolor imaging is required, the imaging assembly 28 may include one or more basic imaging modules, and the direction of the fluorescence in the imaging assembly is shown by the arrow in FIG. 7 . This embodiment includes three basic imaging modules, wherein the first basic forming module 47 , the second basic imaging module 48 , and the third basic imaging module 49 are composed of the same optical elements and have the same structure. Taking the first basic imaging module 47 as an example, it includes a sixth dichroic mirror 50 , a first filter 51 , a second filter 52 , a second lens 53 and a single photon counter 54 . Among them, the function of the sixth dichroic mirror 50 is to separate the fluorescence signal detected in the channel and the fluorescence signal of the subsequent channel, and the transmittance curve is determined by the detected fluorescence spectrum, and the fluorescence spectrum channel of this channel is kept high reflectance (>95%) , to maintain high transmittance (>98%) for the subsequent channel detection fluorescence spectrum; the first filter 51 and the second filter 52 are used to filter stray light signals that do not belong to the detection channel spectrum; the second lens The function of 53 is to focus the collected fluorescence on the photon counter, and the second lens in this embodiment is a doublet lens with a focal length of 125mm. The function of the single-photon counter 54 is to linearly convert the fluorescent signal into an electrical signal according to the number of collected fluorescent photons; the photon counter in this embodiment selects an avalanche light-emitting diode or a photomultiplier tube. The electrical signal generated by the single photon counter 54 is finally read by a computer and restored into a fluorescent image.

The multi-color three-dimensional super-resolution expansion microscope system with a large field of view provided by the invention can realize three-dimensional space large field of view microscopic imaging through a sample stage, a scanning component and an adaptive aberration correction component. Specifically, the sample stage has the ability to move in three dimensions, and the scanning component is a three-galvo mirror system, including a first scanning mirror, a second scanning mirror and a third scanning mirror arranged in sequence along the optical path. By changing the mixed beam at the entrance pupil of the microscope objective The deflection angle of the hybrid beam can achieve two-dimensional high-speed scanning on the focal plane of the microscope objective lens, and the combination of the two can achieve three-dimensional fast scanning; the adaptive aberration correction component can solve the aberration problem caused by deep imaging, while ensuring the resolution Under the premise of the ability to achieve deep imaging, the imaging depth is increased, and the three-dimensional space large field of view microscopic imaging is realized.

The above-mentioned embodiments are used to explain the present invention, rather than limit the present invention. Within the spirit of the present invention and the protection scope of the claims, any modifications and changes made to the present invention all fall into the protection scope of the present invention.

Claims (8)

1. A multi-color three-dimensional super-resolution dilation microscope system having a large field of view, the system comprising: an illumination unit and a detection unit;

the illumination unit comprises a loss light path, an excitation light path and a mixed light path;

the loss light path is sequentially provided with a loss light source, a first light modulator, a relative phase delay assembly, a first reflector, a first telescopic assembly, a phase modulation assembly for performing phase modulation on a loss light beam, a second telescopic assembly, a second reflector, a first 1/2 wave plate and a third telescopic assembly; the relative phase delay component splits the loss light beam into two polarization components which are perpendicular to each other and generates phase delay for the two components to destroy the coherence of the two components;

the excitation light path is sequentially provided with an excitation light source, a second light modulator, a dispersion compensation component and a fourth telescopic component; the dispersion compensation component is used for compensating the dispersion of light with different wavelengths;

the mixed light path is sequentially provided with a first dichroic mirror, a second dichroic mirror, a scanning assembly, a fifth telescopic assembly, a self-adaptive aberration correction assembly, a first 1/4 wave plate, a sixth telescopic assembly, a third reflector, a microscope objective and a sample stage; a beam splitter is arranged on a light path in the sixth telescopic assembly, and the first dichroic mirror combines a light beam of the loss light coming out of the third telescopic assembly and a light beam of the excitation light coming out of the fourth telescopic assembly into a mixed light beam; the scanning assembly is used for changing the deflection angle of the mixed light beam at the entrance pupil of the microscope objective so that the focus of the mixed light beam realizes two-dimensional scanning on the focal plane of the microscope objective; the self-adaptive aberration correction component is sequentially provided with a ninth reflector and a deformable mirror; the mixed light beam reaches the surface of the deformable mirror after being reflected by the ninth reflector, and the wavefront of the mixed light beam is corrected;

the detection unit comprises a scattered light imaging component and a fluorescence imaging component, the mixed light beam irradiates on the sample to generate a scattered signal, and the scattered light imaging component is arranged on a reflection light path of the scattered signal passing through the beam splitter and is used for detecting the scattered signal of the sample; the original path of a fluorescence signal generated by the excitation of the mixed light beam returns to be corrected before the wave front of the deformable mirror, and finally reaches the fluorescence imaging component; the fluorescence imaging component is arranged on a transmission light path of the fluorescence signal passing through the second dichroic mirror and used for detecting the fluorescence signal of the sample.

2. A multi-color three-dimensional super-resolution dilation microscope system with a large field of view according to claim 1, wherein: the system realizes multicolor fluorescence imaging through a second light modulator, a dispersion compensation component and a fluorescence imaging component;

the device comprises an excitation light source, a first light modulator, a second light modulator, an acousto-optic tunable filter, a sample and a control circuit, wherein the excitation light source is a white light laser, the second light modulator is arranged behind the white light laser and used for selecting the wavelength of an excitation light beam and modulating the transmission light intensity of the corresponding wavelength, the wavelength required to be modulated is determined by fluorescent dye used in the sample, and the wavelength comprises 488nm, 590nm and 650 nm; the dispersion compensation component comprises a third 1/2 wave plate, a third polarization beam splitter, a second 1/4 wave plate, a third dichroic mirror, a fourth dichroic mirror and a fifth dichroic mirror which are arranged in sequence; the relative positions of the three dichroic mirrors are determined according to the dispersion value corresponding to the wavelength of the excitation light, namely, the relative positions are determined by the emission sequence corresponding to the excitation light pulses with different wavelengths, in a white light laser, the 650nm excitation light is firstly emitted, then 590nm and finally 488 nm; the three paths of exciting light firstly pass through a third 1/2 wave plate to become vertical polarized light, then are reflected by a third polarization beam splitter, and then pass through a second 1/4 wave plate; then, 488nm excitation light returns through a third dichroic mirror primary reflection path, 590nm excitation light returns through a third dichroic mirror transmission path and a fourth dichroic mirror primary reflection path, 650nm excitation light returns through the third dichroic mirror transmission path, the fourth dichroic mirror transmission path and a fifth dichroic mirror primary reflection path; the three paths of exciting light returned by the original path after reflection pass through the 1/4 wave plate again to be changed into horizontal polarized light, the horizontal polarized light is transmitted by the third polarization beam splitter, and finally is focused on a sample by a microscope objective to generate a fluorescence signal, and the fluorescence signal is collected by the fluorescence imaging component; the fluorescence imaging component comprises a sixth dichroic mirror used for separating a fluorescence signal detected by the current imaging module and a fluorescence signal of a subsequent imaging module, an optical filter used for filtering a stray light signal which does not belong to the outside of a detection channel spectrum, a second lens used for focusing the collected fluorescence on the photon counter, and the photon counter used for linearly converting the fluorescence signal into an electric signal according to the number of the collected fluorescence photons.

3. The system of claim 1, wherein the phase modulation assembly comprises a seventh mirror, a spatial light modulator, a third 1/4 wave plate, a first lens, and an eighth mirror; the first lens is positioned in the middle of the spatial light modulator and the eighth reflector, and the focal length of the first lens is equal to the distance between the first lens and the eighth reflector; the loss light firstly reaches a first phase diagram area of the spatial light modulator, and primary phase modulation is carried out on a horizontal polarization component of the loss light; then, the loss light is reflected by the spatial light modulator, then sequentially passes through a third 1/4 wave plate and a first lens, then returns after being reflected by an eighth reflector, and reaches a second phase diagram area of the spatial light modulator again after passing through the first lens and a third 1/4 wave plate to perform secondary phase modulation on the vertical polarization component of the loss light source; the modulated loss light is focused by a microscope objective to form a focusing light spot, and the light intensity in the focusing light spot is distributed in a hollow manner around the focus; the first phase diagram and the second phase diagram of the spatial light modulator are circular and are positioned in the horizontal central position of the effective area of the whole spatial light modulator, the positions of the two phase diagrams can be exchanged, and the diameter of the two phase diagrams is as large as the effective aperture of the phase modulation component.

4. The multi-color three-dimensional super-resolution expansive microscope system with a large field of view of claim 1, wherein the scanning assembly comprises a first scanning mirror, a second scanning mirror and a third scanning mirror arranged in sequence; the effective aperture of the first scanning mirror and the effective aperture of the scanning component are equal, and the scanning direction is vertical to the second scanning mirror and the third scanning mirror; the first scan mirror scans at a faster frequency than the second and third scan mirrors.

5. The system according to claim 1, wherein the first, second, third, fourth, fifth and sixth telescopic elements each comprise two convex lenses with convex surfaces facing away and being confocal; the device is used for expanding or reducing light beams, collimating and maintaining the conjugate relation of the spatial light modulator, the first scanning mirror, the deformable mirror and the rear focal plane of the microscope objective in the system.

6. A multi-color three-dimensional super-resolution dilation microscope system with a large field of view according to claim 1, wherein: the system ensures the incoherent superposition of two beams of loss light through a relative phase delay component, thereby realizing three-dimensional super-resolution microscopic imaging;

the relative phase delay assembly comprises a second 1/2 wave plate and a first polarization beam splitter which divide the loss light beam into a vertical polarization component and a horizontal polarization component, a fourth reflector which is used for changing the direction of the vertical polarization component, a fifth reflector and a sixth reflector which are used for changing the direction and the optical path of the horizontal polarization component, and a second polarization beam splitter which combines the vertical polarization component and the horizontal polarization component.

7. A multi-color three-dimensional super-resolution dilation microscope system with a large field of view according to claim 1, wherein: the system can be combined with an expansion microscopic technology, and can also solve the problem of aberration caused by mismatching of refractive indexes of an expanded biological sample and an oil immersion objective lens through a self-adaptive aberration correction component.

8. A multi-color three-dimensional super-resolution dilation microscope system with a large field of view according to claim 1, wherein: the system can realize three-dimensional space large-field microscopic imaging through the sample stage, the scanning assembly and the self-adaptive aberration correction assembly;

the scanning component is a three-vibrating mirror system and comprises a first scanning mirror, a second scanning mirror and a third scanning mirror which are sequentially arranged along an optical path, the deflection angle of the mixed light beam at the entrance pupil of the microscope objective is changed, so that the focus of the mixed light beam realizes two-dimensional high-speed scanning on the focal plane of the microscope objective, and the two scanning mirrors are combined to realize three-dimensional rapid scanning; the self-adaptive aberration correction assembly can solve the aberration problem caused by deep imaging, and realizes deep imaging on the premise of ensuring the resolution capability, so that the imaging depth is increased, and further, the three-dimensional space large-field microscopic imaging is realized.

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