CN108303806B - Depth imaging super-resolution microscopic imaging system - Google Patents
Depth imaging super-resolution microscopic imaging system Download PDFInfo
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- CN108303806B CN108303806B CN201810097037.9A CN201810097037A CN108303806B CN 108303806 B CN108303806 B CN 108303806B CN 201810097037 A CN201810097037 A CN 201810097037A CN 108303806 B CN108303806 B CN 108303806B
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Abstract
The invention discloses a deep imaging super-resolution microscope system which comprises a first laser, a first lens, a first dichroic mirror, a second laser, a sixth lens, a phase modulation component, a third dichroic mirror, a deformable reflector, an X-axis scanning galvanometer, a phase compensation component, a Y-axis scanning galvanometer, a vertical light path refraction and rotation matching unit, a second quarter-wave plate, a microscope objective, a sample stage, a horizontal light path refraction and rotation matching unit, a first light filter, a wavefront sensor, a second light filter, a tenth lens and an imaging device. The system compensates the phase change of the fluorescence wavefront by using the two-photon effect, the wavefront sensor and the deformable reflector, and realizes three-dimensional depth imaging while correcting the aberration caused by the sample by combining the movement of the sample stage in the Z-axis direction, so that the imaging quality is higher.
Description
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a depth imaging super-resolution microscopic imaging system.
Background
In a conventional optical microscopic imaging system, due to the diffraction effect of optical components, a light spot formed on a sample after parallel incident illumination light is focused by a microscope objective is not an ideal point but a diffraction light spot with a certain size, and according to an abbe diffraction limit formula, the diameter of the minimum light spot of visible light focusing is about 200 nm. The first proposed STED super-resolution microscopy by german scientist s.w. hel in 1994, which surpassed the diffraction limit and achieved a spatial resolution of 30 nm in 2006, which was an outstanding task that led him to the nobel prize in 2014.
The basic idea of STED super resolution is: the stimulated emission effect is used to reduce the effective fluorescence area, and a typical STED microscope system requires two beams of light to achieve the above purpose, one beam being exciting light and the other beam being loss light. When the exciting light irradiates the fluorescent sample, fluorescent molecules of light in the exciting light focusing light spot are activated, electrons in the fluorescent molecules jump to an excited state, the focusing light spot is formed by combining hollow circular loss light and the exciting light, electrons in the excited state of the overlapped part of the exciting light focusing light spot and the loss light focusing light spot return to a ground state in an excited radiation mode, and other excited electrons in the hollow position of the loss light focusing light spot continue to generate fluorescence outwards in a spontaneous radiation mode to return to the ground state due to the fact that the other excited electrons are not influenced by the loss light. Because the directions and the wavelengths of the fluorescence emitted in the processes of the stimulated radiation and the spontaneous radiation are different, the photons received by the detector after being filtered are generated by the fluorescence sample positioned at the central position of the excitation light focusing spot in an autofluorescence mode. Thus, the light emitting area of the effective fluorescence is reduced, thereby improving the spatial resolution of the system.
In the application of the existing STED super-resolution microscopic imaging system in biomedicine, although super-resolution is realized and the imaging resolution is improved to a certain extent in a Longkou excellent patent document, namely a stimulated emission depletion microscope device, publication No. CN106133580A, the single photon excitation is greatly influenced by scattering, so that the excitation light is not suitable to penetrate through a sample, the defects of serious photobleaching and the like influence the resolution of the system, and the wide application of the system is limited.
With the development of biomedical research, researchers have higher and higher requirements on the resolution and the imaging depth of the STED super-resolution microscopic imaging system. The wavelength is longer when the two-photon excitation is carried out, and the influence of scattering is smaller; in addition, the fluorescent molecules outside the focal plane are not excited, so that more exciting light can reach the focal plane, and compared with single photon excitation, the exciting light excited by two photons can penetrate deeper specimens, and photobleaching and phototoxicity are low, so that the method is suitable for three-dimensional deep living cell imaging, and people begin to combine the two-photon excitation technology with the STED super-resolution microscopic imaging system. In the patent document STED fluorescence microscopy with two-photon excitation, publication No. CN101821607A of s.w.hel et al, single-photon excitation is replaced by two-photon excitation, but the system cannot realize high-resolution imaging of deep tissue due to aberration caused by unevenness of the sample surface and non-uniformity of refractive index distribution inside the sample, which limits its wide application.
Disclosure of Invention
The invention provides a depth imaging super-resolution microscope system aiming at the aberration problem of sample depth direction scanning when the existing super-resolution microscope system realizes three-dimensional imaging of a biological sample structure. The system utilizes the two-photon effect, compensates wavefront phase change brought by aberration by combining with a deformable reflector, corrects the aberration brought by a sample, and realizes three-dimensional super-resolution imaging.
A deep imaging super-resolution microscopic imaging system comprises a first laser, a first lens, a first dichroic mirror, a second laser, a sixth lens, a phase modulation assembly, a third dichroic mirror, a deformable reflector, an X-axis scanning galvanometer, a phase compensation assembly, a Y-axis scanning galvanometer, a vertical light path deflection matching unit, a second quarter-wave plate, a microscope objective, a sample stage, a horizontal light path deflection matching unit, a first light filter, a wavefront sensor, a second light filter, a tenth lens and an imaging device;
excitation light output by the first laser reaches the second dichroic mirror after being expanded by the first lens and transmitted by the first dichroic mirror;
the loss light output by the second laser reaches the second dichroic mirror after being expanded by the sixth lens and phase modulated by the phase modulation component, and is superposed with the exciting light reaching the second dichroic mirror to form superposed light;
the coincident light is transmitted by the third dichroic mirror and the deformable reflector, then is scanned by the X-axis scanning galvanometer, the phase compensation assembly compensates the phase, and the Y-axis scanning galvanometer, finally enters the microscope objective after passing through the vertical light path deflection matching unit and the second quarter-wave plate, forms a focusing light spot with the resolution smaller than the diffraction limit on a focusing plane of the microscope objective, and excites the fluorescence of a sample on the sample stage;
the sample is marked in advance by two kinds of fluorescence, and the sample emits two kinds of fluorescence with different wavelengths after being excited;
a telescopic system for adjusting light beams is arranged between the second dichroic mirror and the third dichroic mirror, between the deformable reflecting mirror and the X-axis scanning galvanometer, between the X-axis scanning galvanometer and the Y-axis scanning galvanometer, and between the phase modulation assembly and the second dichroic mirror;
the fluorescence with two different wavelengths collected by the microscope objective returns along the original path of the incident light path of the coincident light until the fluorescence is incident to a third dichroic mirror, wherein the fluorescence with one wavelength (called as first fluorescence) is reflected by the third dichroic mirror, then is detected by the wavefront sensor after passing through the horizontal light path deflection matching unit and the first optical filter, and the work of the deformable reflector is controlled according to the wavefront information of the wavefront sensor; and the other fluorescence with different wavelengths (called as second fluorescence) passes through the third dichroic mirror, is reflected by the second and first dichroic mirrors, enters the imaging device through the second optical filter and the tenth lens, and then is imaged.
In the invention, the vertical light path deflection matching unit is mainly used for converting the light path positioned on the XY plane into the Z direction so as to realize the observation of a horizontally placed sample and simultaneously realize the mutual matching of light beams among different optical elements and the transmission of images.
Preferably, the vertical optical path deflection matching unit comprises a second lens, a second reflector, a third lens, a third reflector, a fourth lens, a fourth reflector and a fifth lens; the superposed light scanned by the X-axis scanning galvanometer and the Y-axis scanning galvanometer sequentially passes through the second lens, the second reflector, the third lens, the third reflector, the fourth lens, the fourth reflector, the fifth lens and the second quarter wave plate to enter the objective lens.
In the invention, the X, Y-axis scanning galvanometers are used for respectively realizing deflection of the superposed light in the X, Y direction, and the X-axis scanning galvanometer and the Y-axis scanning galvanometer are mutually matched to realize point-by-point scanning of an XY surface of a sample. The positions of the beam and the beam can be changed, for example, the beam can be scanned by the Y-axis scanning galvanometer and then by the X-axis scanning galvanometer.
In the invention, the horizontal light path deflection matching unit is mainly used for realizing the guiding of the light path and the matching of the light beam on the XY plane, further optimizing the construction of the system and improving the compactness of the whole system.
Preferably, the horizontal optical path turning and matching unit includes an eighth lens, an eighth mirror, a ninth lens, a tenth mirror, and an eleventh mirror; and the fluorescence reflected by the third dichroic mirror sequentially passes through the eighth lens, the eighth reflector, the ninth lens, the tenth reflector, the eleventh reflector and the first optical filter and enters the wavefront sensor.
In the present invention, the phase modulation component is used for modulating the phase of the input loss light. Preferably, the phase modulation component comprises a fifth reflector, a spatial light modulator, a seventh lens, a third quarter wave plate and a sixth reflector; the loss light expanded by the sixth lens reaches a corresponding phase diagram area of the spatial light modulator for the first time through a fifth reflector to perform first phase adjustment; and then the loss light after the first phase adjustment sequentially passes through a seventh lens, a third quarter wave plate and a sixth reflector, returns to the third quarter wave plate and the seventh lens after being reflected by the sixth reflector, and finally reaches a corresponding phase diagram area of the spatial light modulator for the second phase adjustment.
Preferably, a first reflecting mirror is disposed between the third dichroic mirror and the deformable reflecting mirror. The coincident light transmitted from the third dichroic mirror is reflected by the first mirror onto the anamorphic mirror.
Preferably, a seventh reflecting mirror is disposed between the phase modulation component and the second dichroic mirror. The loss light modulated by the phase modulation component is reflected to the second dichroic mirror through the seventh reflecting mirror.
Preferably, a fifth mirror is disposed between the sixth lens and the spatial light modulator. After the loss light emitted by the second laser passes through the sixth lens, the loss light is reflected to the spatial light modulator through the fifth reflector to be subjected to primary phase modulation.
In the invention, the spatial light modulator is only sensitive to horizontal polarized light and only can adjust the wave front of the horizontal polarized light in the loss light. Therefore, the phase adjustment of the horizontal polarized light in the loss light is firstly completed by utilizing the fifth reflecting mirror to reflect the loss light to a corresponding phase diagram area of the spatial light modulator; the loss light emitted from the spatial light modulator passes through the quarter-wave plate twice to generate half-wave phase delay, the polarization direction of the original horizontal polarization light is changed into the vertical polarization direction and cannot be influenced by a phase pattern on the spatial light modulator, the polarization direction of the original vertical polarization light is changed into the horizontal polarization direction, the phase adjustment of the vertical polarization light in the loss light is completed, and finally a three-dimensional hollow spherical loss light spot is generated.
Preferably, the phase compensation assembly comprises a half-wave plate and a first quarter-wave plate, the half-wave plate and the first quarter-wave plate are combined to compensate uncertain phase delay in the whole system, and the phase compensation assembly is used for ensuring that light entering the vertical optical path refraction matching unit is linearly polarized light.
In the invention, the first laser is used for generating femtosecond laser, and the second laser is used for generating picosecond laser, thereby providing an environment with high photon density, generating two-photon excitation, reducing scattering influence and realizing depth imaging.
In the invention, the two-photon excitation means that under the condition of an environment with high photon density, two photons with long wavelength can be absorbed by fluorescent molecules at the same time, and after a short so-called excited state life, a photon with shorter wavelength is emitted, so that the two-photon excitation has small scattering influence, photobleaching and phototoxicity, can deeply image tissues, and only occurs at the focus of an objective lens, so that the fluorescence detection efficiency is high.
Preferably, the first laser outputs excitation light having a wavelength of 830 nm, and the second laser outputs loss light having a wavelength of 775 nm.
Preferably, mutually independent telescopic systems are arranged between the second dichroic mirror and the third dichroic mirror, between the deformable reflecting mirror and the X-axis scanning galvanometer, between the X-axis scanning galvanometer and the Y-axis scanning galvanometer, and between the phase modulation assembly and the second dichroic mirror.
In the invention, excitation light output by the first laser reaches the second dichroic mirror after being expanded by the first lens and transmitted by the first dichroic mirror; the loss light output by the second laser reaches the spatial light modulator after being expanded by the sixth lens and reflected by the fifth reflector, the loss light which is modulated for the first time by the spatial light modulator reaches the spatial light modulator again after passing through the seventh lens and the third quarter-wave plate twice, and the loss light which is modulated for the second time by the spatial light modulator reaches the second dichroic mirror after being collimated by the fourth telescope system and reflected by the seventh reflector; the second dichromatic mirror reflects the excitation light, transmits the loss light, so that the excitation light and the loss light are coaxially overlapped, the coincident light enters the second telescopic system for collimation after passing through the first telescopic system for collimation, the third dichromatic mirror transmits, the first reflector reflects and the deformation reflector for collimation, then enters the second reflector after passing through the X-axis scanning galvanometer, the third telescopic system, the half-wave plate, the first quarter-wave plate, the Y-axis scanning galvanometer and the second lens, the coincident light reflected by the second reflector passes through the third lens and then is reflected by the third reflector, the coincident light reflected by the third reflector passes through the fourth lens and then is reflected by the fourth reflector, passes through the fifth lens and the second quarter-wave plate and then enters the microscope objective, and finally, the microscope objective focuses on a sample of the sample stage, and the sample is marked by two kinds of fluorescence in advance to excite fluorescence with two wavelengths;
the fluorescence with two wavelengths is reversely collected by the microscope objective, the fluorescence with two wavelengths collected by the microscope objective returns along the primary path of the incident light path of the coincident light until the fluorescence is incident to the third dichroic mirror, and the fluorescence with one wavelength is detected by the wavefront sensor after being reflected by the third dichroic mirror, the eighth lens, the eighth reflector, the ninth lens, the tenth reflector, the eleventh reflector and the first optical filter; and the fluorescence with the other wavelength passes through the third dichroic mirror, is collimated by the first telescope system, reflected by the second and first dichroic mirrors, and enters the imaging device to realize imaging after passing through the second optical filter and the tenth lens.
In the invention, the first, second, third and fourth telescopic systems respectively comprise two convex surfaces which are arranged in a back-to-back manner and are confocal, and the convex lenses are used for expanding (or contracting) and collimating, so that the diameter of a light beam passing through the telescopic system is increased (or decreased), the divergence angle is smaller, and the light intensity distribution in a cross section perpendicular to the optical axis direction is more uniform and closer to parallel light.
In the invention, the first lens is used for collimating exciting light, the sixth lens is used for collimating lost light, the second and fourth lenses are used for converging recombined light, the third and fifth lenses are used for collimating the recombined light, the eighth lens is used for converging first fluorescence, the ninth lens is used for collimating the first fluorescence, and the tenth lens is used for converging the second fluorescence.
In the invention, the first dichroic mirror is high in transmission for exciting light and high in reflection for second fluorescence. The second dichroic mirror exhibits high reflectance for the excitation light and the second fluorescence and high transmittance for the loss light. The third dichroic mirror shows high transmittance for the coincident light and the second fluorescence, and shows high reflectance for the first fluorescence. The high transmittance refers to the transmittance of more than 98 percent; the high reflectivity means that the reflectivity is more than 98%, specifically 98-99.9%.
In the invention, the second quarter-wave plate is used for converting linear polarization into circular polarization.
In the invention, the deformable reflector is used for correcting the wavefront changes of the fluorescence with two wavelengths generated by the unevenness of the surface of the sample and the nonuniformity of the internal refractive index distribution, and performing phase compensation on the fluorescence with two wavelengths, thereby achieving the purpose of compressing the aberration generated by the sample.
According to the invention, the wavefront sensor measures the wavefront of the first fluorescence, and the difference between the wavefront of the first fluorescence and the wavefront of an ideal plane wave is compared, so that the wavefront change of the first fluorescence caused by the sample is obtained; because the wavefront changes of the sample to the first fluorescence and the second fluorescence are the same, the wavefront changes of the first fluorescence and the second fluorescence can be compensated by driving the deformable mirror to generate opposite phase delays, and the compression of the sample aberration is realized.
In the invention, the first and second optical filters are used for filtering stray light, the first optical filter only allows first fluorescence to pass through, and the second optical filter only allows second fluorescence to pass through.
In the invention, the second reflector, the third lens, the third reflector, the fourth lens, the fourth reflector, the fifth lens, the second quarter-wave plate, the microscope objective and the sample stage are arranged on an XZ plane, so that the sample can be observed conveniently, and other devices are arranged on an XY plane.
Preferably, two ATTO series dyes with excellent light stability and brightness and long service life are selected for the fluorescence, so that the fluorescence loss is reduced, and the imaging fluorescence signal is enhanced.
Preferably, the microscope objective is a special objective lens of model XLPN25XSVMP produced by Olympus, with 25 times magnification, 1.0 numerical aperture and 4mm working distance.
Preferably, the X, Y scanning galvanometer is an optical scanning galvanometer manufactured by Cambridge Technology and having a model number of 6231H.
The sample stage can reciprocate in the Z-axis direction at a set speed so as to realize three-dimensional super-resolution imaging of the sample.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the invention has the advantages that the exciting light is infrared light, the penetrating power is strong, the tissue depth imaging is more facilitated, in addition, the energy of photon wavelength is low, and the photobleaching and phototoxicity to the tissue are low.
2. The invention introduces a wavefront sensor to observe the wavefront phase of a sample in real time, flexibly compensates the wavefront change caused by the sample in cooperation with a deformable reflector, realizes phase compensation, thereby achieving the purpose of compressing the aberration generated by the sample, and can realize three-dimensional super-resolution imaging by combining the movement of the sample stage in the Z-axis direction.
Therefore, compared with the prior art, the system has the advantages that the two-photon effect is utilized, the wavefront sensor and the deformable reflector are utilized to compensate the fluorescent wavefront phase change, the movement of the sample stage in the Z-axis direction is combined, the aberration caused by the sample is corrected, meanwhile, the three-dimensional depth imaging is realized, and the imaging quality is higher.
Drawings
FIG. 1 is a drawing of the present invention: an optical path diagram of one embodiment of a depth imaging super-resolution microscopy system configuration;
wherein: the system comprises a first laser 1, a first lens 2, a first dichroic mirror 3, a second dichroic mirror 4, a first telescopic system 5, a third dichroic mirror 6, a first reflecting mirror 7, a deformable reflecting mirror 8, a second telescopic system 9, an X-axis scanning galvanometer 10, a third telescopic system 11, a half-wave plate 12, a first quarter-wave plate 13, a Y-axis scanning galvanometer 14, a second lens 15, a second reflecting mirror 16, a third lens 17, a third reflecting mirror 18, a fourth lens 19, a fourth reflecting mirror 20, a fifth lens 21, a second quarter-wave plate 22, a microscope objective lens 23, a sample stage 24, a second laser 25, a sixth lens 26, a fifth reflecting mirror 27, a spatial light modulator 28, a seventh lens 29, a third quarter-wave plate 30, a sixth reflecting mirror 31, a fourth telescopic system 32, a seventh reflecting mirror 33, an eighth lens 34, an eighth reflecting mirror 35, a ninth reflecting mirror 36, a ninth lens 37, a ninth reflecting mirror 37, A tenth mirror 38, an eleventh mirror 39, a first filter 40, a wavefront sensor 41, a second filter 42, a tenth lens 43, and an imaging device 44.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.
Fig. 1 shows an optical path diagram of an embodiment of a structure of a depth imaging super-resolution microscope system according to the present invention, the system of the embodiment comprises:
the system comprises a first laser 1, a first lens 2, a first dichroic mirror 3, a second dichroic mirror 4, a first telescopic system 5, a third dichroic mirror 6, a first reflecting mirror 7, a deformable reflecting mirror 8, a second telescopic system 9, an X-axis scanning galvanometer 10, a third telescopic system 11, a half-wave plate 12, a first quarter-wave plate 13, a Y-axis scanning galvanometer 14, a second lens 15, a second reflecting mirror 16, a third lens 17, a third reflecting mirror 18, a fourth lens 19, a fourth reflecting mirror 20, a fifth lens 21, a second quarter-wave plate 22, a microscope objective lens 23, a sample stage 24, a second laser 25, a sixth lens 26, a fifth reflecting mirror 27, a spatial light modulator 28, a seventh lens 29, a third quarter-wave plate 30, a sixth reflecting mirror 31, a fourth telescopic system 32, a seventh reflecting mirror 33, an eighth lens 34, an eighth reflecting mirror 35, a ninth reflecting mirror 36, a ninth lens 37, a ninth reflecting mirror 37, A tenth mirror 38, an eleventh mirror 39, a first filter 40, a wavefront sensor 41, a second filter 42, a tenth lens 43, and an imaging device 44.
The first Laser 1 is a femto YLTM-100 high-power femtosecond fiber Laser manufactured by Anyang Laser company, and the second Laser 25 is a MENDOCINO picosecond Laser manufactured by Calmar Laser company.
The first laser 1 outputs excitation light and the second laser 25 outputs loss light; the excitation light reaches the second dichroic mirror 4 after being expanded by the first lens 2 and transmitted by the first dichroic mirror 3; the loss light is expanded by the sixth lens 26 and reflected by the fifth mirror 27 to reach a phase diagram area corresponding to the spatial light modulator 28, the phase adjustment of the horizontal polarized light in the loss light is firstly completed, the loss light emitted from the spatial light modulator 28 passes through the third quarter-wave plate 30 twice by the sixth mirror 31 (the loss light emitted from the spatial light modulator 28 is firstly focused by the seventh lens 29, reflected by the sixth mirror 31 after passing through the third quarter-wave plate 30 and the seventh lens 29 in sequence, the reflected light reaches the phase diagram area corresponding to the spatial light modulator 28 again by the third quarter-wave plate 30 and the seventh lens 29), the phase delay of half-wave is generated, the polarization direction of the original horizontal polarized light is changed into the vertical polarization direction, which is not influenced by the phase diagram on the spatial light modulator 28, the polarization direction of the original vertical polarized light is changed into the horizontal polarization direction, the phase adjustment of the vertical polarized light in the loss light is completed, a three-dimensional hollow spherical loss light is finally generated, and the loss light modulated by the spatial light modulator 28 reaches the second dichroic mirror 4 after being collimated by the fourth telescopic system 32 and reflected by the seventh reflecting mirror 33; the second dichroic mirror 4 reflects exciting light, transmits loss light, so that the exciting light and the loss light are coaxially overlapped, the superposed light enters a second telescope system 9 for collimation while adjusting the beam size after being expanded and collimated by a first telescope system 5, transmitted by a third dichroic mirror 6, reflected by a first reflecting mirror 7 and a deformable reflecting mirror 8, then is expanded and collimated by an X-axis scanning vibrating mirror 10 and a third telescope system 11, and then is combined by a half-wave plate 12 and a first quarter-wave plate 13 to compensate uncertain phase delay in the whole system, so that the superposed light entering a vertical light path refraction and rotation matching unit is ensured to be linearly polarized light, the superposed light after phase compensation is incident on a second quarter-wave plate 22 after being converged by a Y-axis scanning vibrating mirror 14, a second lens 15, reflected by a third reflecting mirror 16, collimated by a third lens 17, reflected by a third reflecting mirror 18, converged by a fourth lens 19, reflected by a fourth reflecting mirror 20 and collimated by a fifth lens 21, the fluorescence passes through the second quarter-wave plate 22 and enters the microscope objective 23, and finally the microscope objective 23 focuses the fluorescence on a sample on the sample stage 24 to excite the fluorescence, and the sample is marked by two kinds of fluorescence and emits two kinds of fluorescence with different wavelengths after excitation; the two kinds of fluorescence with different wavelengths are reversely collected by the microscope objective 23, the two kinds of fluorescence with different wavelengths collected by the microscope objective 23 return along the original path of the incident light path of the coincident light until the fluorescence enters the third dichroic mirror 6, wherein the fluorescence with one wavelength is reflected by the third dichroic mirror 6, converged by the eighth lens 34, reflected by the eighth reflector 35, reflected by the ninth reflector 36, collimated by the ninth lens 37, reflected by the tenth reflector 38, reflected by the eleventh reflector 39 and filtered by the first optical filter 40 to enter the wavefront sensor 41, the wavefront sensor 41 directly measures the wavefront phase of the sample, and the deformable reflector 8 is driven to generate opposite phase delay to compensate the wavefront changes of the fluorescence with two different wavelengths; the fluorescence with another wavelength compensated by the wave front change passes through the third dichroic mirror 6, is collimated by the first telescopic system 5, reflected by the second dichroic mirror 4 and the first dichroic mirror 3, filtered by the second optical filter 42 to remove stray light, and converged by the tenth lens 43, and then enters the imaging device 44 to realize imaging of compression aberration.
In this embodiment, the deformable mirror 8 is used to correct the change of the fluorescence wavefront of two wavelengths generated by the unevenness of the sample surface and the unevenness of the refractive index distribution inside the sample, and perform phase compensation on the fluorescence of two wavelengths, thereby achieving the purpose of compressing the aberration generated by the sample.
The second reflector 16, the third lens 17, the third reflector 18, the fourth lens 19, the fourth reflector 20, the fifth lens 21, the second quarter-wave plate 22, the microscope objective 23 and the sample stage 24 are in an XZ plane, so that the sample can be observed conveniently, and other devices are arranged in an XY plane. In this embodiment, the microscope objective 23 may be a special objective of XLPN25XSVMP available from olympus, with a magnification of 25 times, a numerical aperture of 1.0, and a working distance of 4 mm.
In this embodiment, the X-axis scanning galvanometer 10 and the Y-axis scanning galvanometer 14 are used to respectively realize the deflection of the superposed light in the direction X, Y, and the X-axis scanning galvanometer and the Y-axis scanning galvanometer are matched with each other to realize the point-by-point scanning of the XY surface of the sample. The scanning galvanometer is an optical scanning galvanometer of model 6231H produced by Cambridge Technology, and the line scanning width is 15 mm.
In this embodiment, the sample stage may reciprocate in the Z-axis direction at a set speed, and after the microscopic imaging system of the present invention completes imaging of the current position of the sample, the sample stage is moved to start imaging of the next position of the sample, and finally three-dimensional super-resolution imaging of the sample is achieved.
In this example, the samples were previously calibrated with two fluorescent dyes, ATTO647N and ATTO 594.
In this embodiment the wavefront sensor 41 may be implemented as a Shack-Hartmann sensor.
The imaging device 44 in this embodiment may be implemented as an Avalanche Photodiode (APD) of LY3056480 available from Excelitas corporation.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and those skilled in the art may make several modifications and improvements without departing from the principle of the present invention, which should also be regarded as the protection scope of the present invention.
Claims (9)
1. A deep imaging super-resolution microscopic imaging system is characterized by comprising a first laser, a first lens, a first dichroic mirror, a second laser, a sixth lens, a phase modulation assembly, a third dichroic mirror, a deformable reflector, an X-axis scanning galvanometer, a phase compensation assembly, a Y-axis scanning galvanometer, a vertical light path deflection matching unit, a second quarter-wave plate, a microscope objective, a sample stage, a horizontal light path deflection matching unit, a first light filter, a wavefront sensor, a second light filter, a tenth lens and an imaging device;
excitation light output by the first laser reaches the second dichroic mirror after being expanded by the first lens and transmitted by the first dichroic mirror;
the loss light output by the second laser reaches the second dichroic mirror after being expanded by the sixth lens and phase modulated by the phase modulation component, and is superposed with the exciting light reaching the second dichroic mirror to form superposed light;
the coincident light is transmitted by the third dichroic mirror and the deformable reflector, then is scanned by the X-axis scanning galvanometer, the phase compensation assembly compensates the phase and the Y-axis scanning galvanometer, finally enters the microscope objective after passing through the vertical light path deflection matching unit and the second quarter-wave plate, forms a focusing light spot with the diameter smaller than the diffraction limit on the focusing plane of the microscope objective, and excites the fluorescence of the sample on the sample stage;
the sample is marked by two kinds of fluorescence and emits fluorescence with two wavelengths after being excited;
the fluorescence with two wavelengths collected by the microscope objective returns along the primary path of the incident light path of the coincident light until the fluorescence is incident to a third dichroic mirror, wherein the fluorescence with one wavelength is reflected by the third dichroic mirror, then is detected by the wavefront sensor after passing through the horizontal light path deflection matching unit and the first optical filter, and the deformable reflector is controlled to work according to the wavefront information of the wavefront sensor; the fluorescence with the other wavelength passes through the third dichroic mirror, is reflected by the second and first dichroic mirrors, enters the imaging device through the second optical filter and the tenth lens, and is imaged;
and a telescopic system for adjusting light beams is arranged between the second dichroic mirror and the third dichroic mirror, between the deformable reflecting mirror and the X-axis scanning vibration mirror, between the X-axis scanning vibration mirror and the Y-axis scanning vibration mirror, and between the phase modulation assembly and the second dichroic mirror.
2. The depth imaging super-resolution microscopy imaging system according to claim 1, wherein the vertical optical path deflection matching unit comprises a second lens, a second reflector, a third lens, a third reflector, a fourth lens, a fourth reflector, a fifth lens;
the superposed light scanned by the X-axis scanning galvanometer and the Y-axis scanning galvanometer sequentially passes through the second lens, the second reflector, the third lens, the third reflector, the fourth lens, the fourth reflector, the fifth lens and the second quarter wave plate to enter the objective lens.
3. The depth imaging super-resolution microscopy imaging system according to claim 1, wherein the horizontal optical path deflection matching unit comprises an eighth lens, an eighth reflector, a ninth lens, a tenth reflector and an eleventh reflector;
and the fluorescence reflected by the third dichroic mirror sequentially passes through the eighth lens, the eighth reflector, the ninth lens, the tenth reflector, the eleventh reflector and the first optical filter and enters the wavefront sensor.
4. The depth imaging super-resolution microscopy imaging system according to claim 1, wherein the phase modulation assembly comprises a fifth mirror, a spatial light modulator, a seventh lens, a third quarter wave plate, a sixth mirror;
the loss light expanded by the sixth lens reaches a corresponding phase diagram area of the spatial light modulator for the first time through a fifth reflector to perform first phase adjustment; and then the loss light after the first phase adjustment sequentially passes through a seventh lens, a third quarter wave plate and a sixth reflector, returns to the third quarter wave plate and the seventh lens after being reflected by the sixth reflector, and finally reaches a corresponding phase diagram area of the spatial light modulator for the second phase adjustment.
5. The depth imaging super-resolution microscopy imaging system of claim 1, wherein the phase compensation assembly comprises a half-wave plate and a first quarter-wave plate, the half-wave plate and the first quarter-wave plate in combination compensating for phase retardation introduced by a dichroic mirror in the system.
6. The depth imaging super-resolution microscopy imaging system according to claim 1, wherein a first mirror is disposed between the third dichroic mirror and the deformable mirror.
7. The depth imaging super-resolution microscopy imaging system according to claim 1, wherein a seventh mirror is disposed between the phase modulation assembly and the second dichroic mirror.
8. The depth imaging super-resolution microscopy imaging system of claim 1, wherein the first laser is configured to generate a femtosecond laser and the second laser is configured to generate a picosecond laser.
9. The depth imaging super-resolution microscopy imaging system as claimed in claim 1 or 8, wherein the first laser outputs excitation light at a wavelength of 830 nm and the second laser outputs depletion light at a wavelength of 775 nm.
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| CN110376135B (en) * | 2019-08-08 | 2020-07-21 | 中国人民解放军军事科学院国防科技创新研究院 | Terahertz super-resolution microscopic imaging system |
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| CN115015200A (en) * | 2022-06-13 | 2022-09-06 | 华侨大学 | Nano-precision fluorescence imaging device and method based on spatial light modulation |
| CN114859577B (en) * | 2022-07-07 | 2022-10-28 | 浙江大学 | Self-adaptive optical correction system based on biological tissue imaging |
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