CN205003084U - Super -resolution imaging system - Google Patents

Abstract

The utility model discloses a super-resolution imaging system, which comprises a first laser, a second laser, a first half-wave plate, a first pulse beam splitter, a spectrometer, a first lens group, a glass rod, a polarization-maintaining optical fiber, a space light Modulator, first objective lens, mirror group, second lens group, mirror, single-mode fiber, second half-wave plate, retroreflector, mirror, first dichroic mirror, second dichromatic mirror, scanning system, Quarter slide, space objective lens, second pulse splitter and photomultiplier tube, use coherent adaptive optics technology for aberration correction to improve the spatial resolution of the STED super-resolution microscopy imaging system, and solve the problem caused by biological samples Aberrations caused by uneven surface and uneven refractive index distribution in the sample; improved spatial resolution.

Description

A super-resolution imaging system

technical field

The utility model belongs to the field of optical microscopic imaging, and more specifically relates to a method and a system for improving the spatial resolution of stimulated emission depletion (Stimulated Emission Depletion, STED) super-resolution microscopic imaging.

Background technique

In a conventional optical microscopic imaging system, due to the diffraction effect of optical components, the light spot formed on the sample after the parallel incident illumination light is focused by the microscopic objective lens is not an ideal spot, but a spot with a certain size. Diffraction spot, according to Abbe's law proposed by German physicist Ernst-Abbe, the diameter of the smallest spot that visible light can focus is one-third of the wavelength of the light wave, about 200nm. In 1994, German scientist S.W.Hell first proposed STED super-resolution microscopy imaging technology, which surpassed the diffraction limit and achieved a spatial resolution of 30nm in 2006. This outstanding work won him the Nobel Prize in Chemistry in 2014 .

The basic idea of STED super-resolution is to use the stimulated emission effect to reduce the effective fluorescent light-emitting area. A typical STED microscope system requires two beams of light, one for excitation light and the other for depletion light. When the excitation light irradiates the fluorescent sample, the fluorescent molecules within the diffraction spot range will be excited, and the electrons in it will transition to the excited state, and then the circular depletion light is superimposed on the excitation light, and the depletion light makes The electrons in the excited state of the overlapping part return to the ground state in the form of stimulated radiation, and the other excited state electrons located in the center of the laser spot are not affected by the depletion light, and continue to fluoresce outward in the form of spontaneous emission to return to the ground state. Since the directions and wavelengths of fluorescence emitted during stimulated radiation and spontaneous radiation are different, the photons received by the detector after filtering are all produced by the fluorescent sample located in the center of the excitation light spot through autofluorescence. In this way, the light-emitting area of the effective fluorescence is reduced, thereby improving the spatial resolution of the system.

At present, in the application of STED super-resolution microscopy imaging system in biomedicine, the resolution and imaging depth of the system are greatly reduced due to the aberrations caused by the unevenness of the sample surface and the inhomogeneity of the refractive index distribution inside the sample. its wide application.

Utility model content

Aiming at the defects of the prior art, the purpose of this utility model is to use coherent optical adaptive optics technology (CoherentOpticalAdaptiveTechnique, COAT) to correct the aberration to improve the spatial resolution of the STED super-resolution microscopic imaging system, aiming to solve the problems caused by the biological sample surface Aberrations introduced by unevenness and inhomogeneous refractive index distribution within the sample.

The utility model provides a super-resolution imaging system, comprising: a first laser Laser1, a second laser Laser2, a first half-wave plate, a first pulse splitter PS1, a spectrometer SPEC, and a first lens group (L1, L2) , glass rod GR, polarization maintaining fiber Fiber1, spatial light modulator SLM, first objective lens L3, mirror group, second lens group (L4, L5), mirror M4, single-mode fiber Fiber2, second half-wave plate, Retroreflector RR, reflector M5, first dichroic mirror DM1, second dichroic mirror DM2, scanning system Scanner, quarter slide, space objective lens L6, second pulse splitter PS2 and photomultiplier tube PMT, The first laser, Laser1, is used to generate femtosecond laser; the second laser, Laser2, is used to generate picosecond laser; the first half-wave plate is arranged on the exit optical path of the first laser, Laser1, to make the femtosecond laser linearly polarized And adjust the direction of the linearly polarized light; the first pulse splitter PS1 is used to divide the femtosecond laser adjusted by the first half-wave plate into two paths, a part of the light is transmitted into the spectrometer, and the other part of the light is reflected as depleted light; the spectrometer The SPEC is set on the first exit light path of the first pulse splitter for real-time monitoring of the wavelength of the depleted light; the first lens group (L1, L2) is set on the second path of the first pulse splitter The exit light path is used to adjust the depleted light; the glass rod GR is used to widen the adjusted depleted light so that the pulse width of the depleted light is 1 picosecond; the polarization-maintaining fiber Fiber1 is used to adjust the pulse width The depleted light of 1 picosecond is further broadened, so that the pulse width of the depleted light is 200 picoseconds; the spatial light modulator SLM is used to generate the annular spot and is used as an aberration correction system; the first objective lens L3 is used for pulse The depleted light with a width of 200 picoseconds is adjusted so that the diameter of its light spot is equal to the width of the liquid crystal panel in the spatial light modulator (SLM); the reflector group is arranged on the outgoing light of the first objective lens L3 and the spatial light modulator Between the incident light, it is used to adjust the outgoing light of the first objective lens L3 so that it enters the spatial light modulator SLM at an incident angle of 3°-9°, and the second lens group (L4, L5) is used to The outgoing light of the spatial light modulator SLM is transmitted; the mirror M4 is arranged on the transmission light path of the second lens group, and its incident light is the outgoing light of the second lens group; the single-mode fiber Fiber2 is arranged on the second laser Laser2 The outgoing optical path is used to adjust the mode of the picosecond laser; the second half-wave plate is arranged on the outgoing optical path of the first laser Laser1, which is used to make the picosecond laser a linearly polarized light and adjust the direction of the linearly polarized light; The retroreflector RR is used to reflect the picosecond laser adjusted by the second half-wave plate to control the delay time between the excitation light and the depletion light pulse; the incident light of the reflector M5 is that of the retroreflector RR Reflected light, the first incident light of the first dichroic mirror DM1 is the depletion light reflected by M4, and the second incident light is the excitation light reflected by M5, It is used to reflect the depletion light, transmit the excitation light, and adjust the directions of the depletion light and the excitation light so that they overlap; the second dichroic mirror DM2 is set on the exit light path of the first dichroic mirror, and is used for the excitation light and the depletion light are transmitted, and the fluorescence is reflected; the scanning system Scanner is set on the exit light path of the second dichroic mirror, and is used for synchronously scanning the overlapping excitation light and depletion light to realize area scanning ; A quarter of the glass slide is used to adjust the scanned laser light so that it is circularly polarized light; the space objective lens L6 is used to focus the circularly polarized light and collect the fluorescent signal fed back from the sample; the second pulse splitter PS2 is used to divide the fluorescence reflected by the second dichroic mirror DM2 into two parts, one part of the light is reflected into the adaptive optics AO aberration correction system for real-time correction of the system aberration; the other part of the light is transmitted; the photomultiplier tube The PMT is used to amplify the fluorescence transmitted by the second pulse beam splitter and perform super-resolution imaging. Coherent adaptive optical aberration correction COAT system is composed of spatial light modulator SLM and adaptive optics system AO, which is used to correct the aberration of the system in real time and improve the spatial resolution and imaging depth of the STED system.

Still further preferably, the incident angle of the spatial light modulator SLM is 6°.

Still further preferably, it further includes an optical isolator FI arranged between the first laser Laser1 and the first half-wave plate and used to protect the laser.

Still further preferably, the first laser and the second laser are triggered synchronously, and the interval between the peaks of the two laser pulses is kept at 160 ps-200 ps.

Even more preferably, the interval is 180ps.

Still further preferably, the spiral gray-scale phase map for generating ring light and the gray-scale phase map for coherent adaptive optical aberration correction are simultaneously loaded on the spatial light modulator (SLM). Among them, the spiral gray-scale phase image is used to generate a circular depletion light spot, which is superimposed with the excitation light to form a spot beyond the diffraction limit, and then a super-resolution image is formed by scanning, while the coherent adaptive optical aberration correction system is used to correct The aberrations caused by the unevenness of the sample surface and the inhomogeneity of the refractive index distribution inside the sample make our super-resolution imaging system have higher resolution and deeper imaging depth.

Further preferably, the first pulse splitter PS1 splits the laser light into two paths according to 9:1, a small part of the light is transmitted into the spectrometer, and most of the light is reflected as depletion light. The second pulse splitter PS2 divides the fluorescence into two parts according to 9:1, one part of the light is reflected into the adaptive optics AO aberration correction system for real-time correction of the system aberration; the other part of the light is transmitted.

The utility model realizes the control of coherence enhancement and coherence weakening of the two beams of coherent light by regulating the phase of one beam of light in the two beams of coherent light, and realizes the aberration correction of the microscopic system, thereby improving the high STED super-resolution display. The spatial resolution of the micro-imaging system can solve the problem of poor image quality caused by the aberrations introduced by the uneven surface of the biological sample and the uneven distribution of the refractive index in the sample when the existing super-resolution imaging system is imaging deep biological cells.

Description of drawings

Fig. 1 is the optical path structure diagram of the super-resolution imaging system provided by the implementation of the utility model;

Figure 2 is a schematic diagram of exceeding the diffraction limit after depletion light and excitation light overlap;

Fig. 3 is the time interval between the two pulse peaks of the excitation light pulse and the depletion light;

Figure 4 is the Confocal image and STED super-resolution image of 170nm fluorescent beads obtained by the system;

Fig. 5 is the aberration correction of the light spot in the scattering sample by using a separate COAT aberration correction system.

detailed description

In order to make the purpose, technical solutions and advantages of the utility model clearer, the utility model will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the utility model, and are not intended to limit the utility model.

A method and system for improving the spatial resolution of STED super-resolution imaging provided by the utility model belong to a stimulated emission depletion (STED) super-resolution microscopic imaging system and a new method for improving the spatial resolution of the system, which can solve the problem of existing super-resolution When the imaging system is imaging deep biological cells, the image quality is poor due to aberration.

The embodiment of the utility model is achieved in this way, a super-resolution imaging system comprising:

A femtosecond laser (Laser1) for generating depletion light;

Picosecond laser (Laser2), used to generate excitation light;

Optical isolator (FI), used to protect the laser;

A glass rod (GR) to stretch the depleted light of the femtosecond pulse to a pulse width of about 1 picosecond;

100 meters of polarization-maintaining fiber (Fiber1), used to further broaden the depleted light so that its pulse width reaches about 200 picoseconds;

A spectrometer (SPEC) for real-time monitoring of relevant parameters such as the wavelength of the depleted light;

Spatial light modulator (SLM), in this system, the spatial light modulator has two purposes, one is to act as the function of the spiral phase plate, used to generate a ring-shaped spot, and the other is used for the aberration correction system;

Single-mode fiber (Fiber2), used to adjust the mode of the laser emitted from the picosecond laser;

Retroreflector (RR) to control the delay time between the excitation and depletion light pulses;

Scanning system (Scanner), used for synchronous scanning of overlapping excitation light and depletion light to realize area scan;

A high numerical space objective lens (L6) is used to focus the overlapping excitation light and depletion light while collecting fluorescence signals; specifically, a Leica objective lens with a numerical aperture of 1.4 can be used.

A photomultiplier tube (PMT) for amplifying the fluorescent signal;

All half-wave plates in this paper ( ), are used to ensure that the laser in the optical path is linearly polarized, and at the same time adjust the direction of the linear polarization;

One quarter of the slide, so that the laser light entering the objective lens is circularly polarized light;

The first pulse splitter (PS1) is used to divide the femtosecond laser into two parts (9:1), a small part of the light is transmitted into the spectrometer, and most of the light is reflected as depletion light;

The second pulse splitter (PS2) is used to divide the collected fluorescence signal into two parts (9:1), and a small part of the light is reflected into the adaptive optics (AO) aberration correction system to correct the system aberration Real-time correction, most of the transmission enters the PMT for super-resolution imaging;

The first dichroic mirror (DM1) is used to reflect the depletion light (780nm) and transmit the excitation light (635nm). At the same time, it can also fine-tune the direction of the depletion light so that the depletion light and the excitation light can overlap well;

The second dichroic mirror (DM2) is used to transmit excitation light and depletion light and reflect fluorescence;

The coherent adaptive optical aberration correction (COAT) system is composed of a spatial light modulator (SLM) and an adaptive optics system (AO), which is used to correct the aberration of the system in real time and improve the space of the STED system of the utility model. resolution and imaging depth;

When working, the excitation light source is turned on first, and then the depletion light source is turned on. Our method is to use the control system of the femtosecond laser to trigger the femtosecond laser and the picosecond laser synchronously, and it is best to keep the peak value between the two laser pulses. The interval between them is 160ps-200ps, preferably 180ps, so as to ensure that the depletion light returns the excited state electrons generated by the excitation light to the ground state in the form of stimulated radiation more cleanly. At the same time, on the spatial light modulator, we will simultaneously load the spiral gray-scale phase image for ring light generation and the gray-scale phase image for coherent adaptive optical aberration correction. During the experiment, these two gray-scale phase images can be used Work at the same time without interfering with each other. The spiral gray-scale phase map is used to generate a circular depletion light spot, which is superimposed with the excitation light to form a spot beyond the diffraction limit, and then a super-resolution image is formed by scanning, while the coherent adaptive optical aberration correction system is used to correct the sample surface The aberrations generated by the unevenness and the inhomogeneity of the refractive index distribution inside the sample make our super-resolution imaging system have higher resolution and deeper imaging depth.

In order to make the purpose, technical solutions and advantages of the utility model clearer, the utility model will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the utility model and not limit the utility model.

The realization of the utility model is described in detail below in conjunction with the embodiments.

As shown in FIG. 1 , it is an optical path system diagram of STED super-resolution microscopic imaging based on COAT aberration correction technology provided by the embodiment of the utility model. From the figure, we can see that the system has two laser beams, which are depletion light (Laser1 wavelength is 780nm) and excitation light (Laser2 wavelength is 635nm). The depletion light first passes through the optical isolator (FI), and the optical isolation The main function of the optical isolator FI here is to prevent the reflected light generated in the optical path from adversely affecting the laser. The optical isolator FI behind The half-wave plate is used to adjust the polarization characteristics of the depleted light to ensure that the depleted light entering the optical system is linearly polarized light. PS1 is a pulse beam splitter, which divides the laser into two beams (9:1). The light is reflected into the follow-up optical path, while a small part of the light is transmitted into the spectrometer (SPEC). The spectrometer SPEC mainly monitors the laser parameters output by the laser in real time, and the reflected light is adjusted by the lens group (L1, L2). The light spot of the size enters the glass rod (GR). The main function of the glass rod here is to broaden the femtosecond depletion pulse light. In the 100-meter-long polarization-maintaining fiber (Fibre1), the main function is to use the fiber to further broaden the pulsed depleted light, and finally obtain the depleted light with a pulse width of 200 picoseconds. The depleted pulsed light adjusted by the polarization-maintaining fiber Fibre1, Then adjust the spot through a suitable objective lens (L3), so that the size and diameter of the spot are consistent with the width of the spatial light adjustment (SLM), and then after a series of mirrors (M1, M2, M3) adjustments, with 3 °～9° (preferably 6°) incident angle enters the spatial light modulator SLM, the role of the spatial light modulator SLM here is mainly two, one is to generate the spiral phase gray scale image, so that the depleted light after reflection It is a hollow circular ring-shaped spot, and another function is to form a COAT aberration correction system with the adaptive optics system (AO) behind to correct the aberration of the depleted light to improve the spatial resolution and imaging depth of STED imaging. The depleted light reflected by the SLM passes through the lens group (L4 and L5) to adjust the spot, enters the scanning system Scanner with an appropriate spot size, and then enters the quarter slide ( ), convert linearly polarized light into circularly polarized light, and finally enter the sample through the objective lens adjustment.

Among them, the size of the light spot should be determined according to the selected galvanometer. The so-called appropriate spot size means that the light spot is just aligned with the center of the two mirrors of the galvanometer, and there is a quarter of a mirror from the edge of the light spot to the edge of the mirror. Width (because the X, Y vibrating surfaces of the vibrating mirror are rectangular mirrors, so the width of the rectangle is mainly discussed here), that is, the spot size should occupy half of the center of the mirror.

The excitation light is coupled into the single-mode fiber Fiber2 by the picosecond laser Laser2. After adjusting the mode of the excitation light, it passes through The half-wave plate adjusts the linear polarization state of the excitation light, and then adjusts the mirrors (RR and M5) and the dichromatic mirrors (DM1 and DM2), and overlaps the depletion light at the scanning system Scanner, the mirror M5 and the dichromatic mirror The mirror DM1 can fine-tune the excitation light and the depletion light at a small angle to ensure that the two beams are highly coincident, as shown in Figure 2. In order to achieve super-resolution imaging in the STED super-resolution system, it is necessary to maintain a time interval of about 180 picoseconds between the pulse peaks of the excitation light and the depletion light, as shown in Figure 3. During the experiment, in order to maintain Stable synchronization, and the generation of such a pulse time interval, we implement the control system of the laser Laser1 to simultaneously trigger the laser Laser2 through the external wire, and the time interval of 180 picoseconds is changed by changing the length of the external trigger cable and adjusting the excitation light path. The position of the retroreflector RR is realized (the length of the external trigger cable is linearly related to the time interval, the longer the length, the longer the time interval; for example, the speed of light is 3*10 ⁸ m/s, after conversion, it can be known that every moving The time delay that can be changed by one meter is 3.333 nanoseconds). After the overlapping excitation light and depletion light are focused by the objective lens, the fluorescent sample is excited, and the fluorescent signal generated by the sample is collected by the objective lens, returns along the original optical path, is reflected at the dichroic mirror DM2, and passes through the beam splitter (9:1 ) and then divided into two beams of light, most of the light is transmitted and then coupled into the multimode fiber (because the fiber port of the single mode fiber is very small, it can play the role of a small hole), and then the multimode fiber is introduced into the photomultiplier tube PMT , the signal is amplified, and finally imaged on the computer, a small part of the light enters the AO system after reflection, and forms a coherent adaptive optical aberration correction system together with the previous SLM, which can correct the aberration introduced by biological samples in real time , thereby improving the spatial resolution and imaging depth of the STED super-resolution system.

In the experiment, we use fluorescent beads with a diameter of 170nm as the experimental sample. We set an electronically controlled baffle on the optical path of the depletion light (between M4 and DM1). When the baffle is closed, the baffle will block the depletion light. , only the excitation light enters the objective lens, which can be regarded as Confocal imaging at this time. When the baffle is opened, the depletion light will overlap with the excitation light on the sample to form a STED super-resolution imaging, as shown in Figure 4. For our STED The 170nm fluorescent bead image collected by the super-resolution imaging system (COAT aberration correction is not turned on), comparing the Confocal image and the STED image of the fluorescent bead in the figure, we can clearly see that after the depletion of light, the point of the excitation light There is indeed a significant reduction in the extension function. Although the STED super-resolution imaging system has achieved a spatial resolution of 30nm as early as 2006, in biological samples, due to the unevenness of the sample surface and the inhomogeneity of the refractive index resolution inside the sample, the STED super-resolution The system introduces a large aberration, which reduces the spatial resolution and imaging depth of the system. In order to overcome this problem, we propose to introduce the COAT aberration correction system in the STED super-resolution imaging system, as shown in Figure 5, for us to use The separate COAT aberration correction system realizes the aberration correction of the spot in the scattering sample. Therefore, after we introduce COAT in this system, it should be able to overcome the aberration introduced in the system and realize the improvement of STED super-resolution spatial resolution and imaging depth. .

Those skilled in the art can easily understand that the above descriptions are only preferred embodiments of the present utility model, and are not intended to limit the present utility model. Any modifications, equivalent replacements and modifications made within the spirit and principles of the present utility model Improvements and the like should all be included within the protection scope of the present utility model.

Claims (8)

1. A super-resolution imaging system, characterized in that, comprising: The first laser, Laser1, is used to generate femtosecond laser; The second laser Laser2 is used to generate picosecond laser; The first half-wave plate is arranged on the outgoing light path of the first laser Laser1, and is used to make the femtosecond laser be linearly polarized light and adjust the direction of the linearly polarized light; The first pulse beam splitter is used to divide the femtosecond laser light adjusted by the first half-wave plate into two paths, a part of the light is transmitted into the spectrometer, and the other part of the light is reflected as depletion light; A spectrometer, arranged on the first outgoing optical path of the first pulse splitter, for real-time monitoring of the wavelength of the depleted light; The first lens group is arranged on the second outgoing light path of the first pulse beam splitter, and is used to adjust the depleted light; a glass rod, used to stretch the adjusted depleted light so that the pulse width of the depleted light is 1 picosecond; A polarization-maintaining fiber is used to further broaden the depleted light with a pulse width of 1 picosecond, so that the pulse width of the depleted light is 200 picoseconds; Spatial light modulator, used to generate the annular light spot and as an aberration correction system; The first objective lens L3 adjusts the depleted light with a pulse width of 200 picoseconds so that the diameter of its spot is equal to the width of the liquid crystal panel in the spatial light modulator; The reflective mirror group is arranged between the outgoing light of the first objective lens L3 and the incident light of the spatial light modulator, and is used to adjust the outgoing light of the first objective lens L3 so that it has an angle of 3°-9° The incident angle enters the spatial light modulator, The second lens group is used to transmit the outgoing light of the spatial light modulator SLM; The mirror M4 is arranged on the transmitted light path of the second lens group, and its incident light is the outgoing light of the second lens group; A single-mode optical fiber is arranged on the outgoing optical path of the second laser Laser2, and is used to adjust the mode of the picosecond laser; The second half-wave plate is arranged on the outgoing optical path of the first laser Laser1, and is used to make the picosecond laser light linearly polarized and adjust the direction of the linearly polarized light; a retroreflector, used to reflect the picosecond laser adjusted by the second half-wave plate, and control the delay time between the excitation light and the depletion light pulse; The reflector M5, whose incident light is the reflected light of the retroreflector; The first dichroic mirror, whose first incident light is the depletion light reflected by the reflector M4, and whose second incident light is the excitation light reflected by the reflector M5, is used to reflect the depletion light , transmitting the excitation light, and adjusting the directions of the depletion light and the excitation light to overlap; a second dichroic mirror, arranged on the outgoing light path of the first dichroic mirror, for transmitting the excitation light and the depletion light, and reflecting the fluorescence; A scanning system, arranged on the outgoing light path of the second dichroic mirror, for synchronously scanning the overlapping excitation light and depletion light to realize area scanning; A quarter glass slide is used to adjust the scanned laser light so that it is circularly polarized light; The space objective lens L6 is used to focus the circularly polarized light and collect the fluorescent signal fed back from the sample; The second pulse beam splitter is used to divide the fluorescence reflected by the second dichroic mirror into two parts, one part of the light is reflected into the adaptive optical aberration correction system, and is used for real-time correction of the system aberration; the other part of the light is transmitted; The photomultiplier tube is used for amplifying the fluorescence transmitted by the second pulse beam splitter and performing super-resolution imaging. 2. The super-resolution imaging system according to claim 1, wherein the incident angle of the spatial light modulator SLM is 6°. 3. The super-resolution imaging system according to claim 1, further comprising an optical isolator arranged between the first laser Laser1 and the first half-wave plate and used to protect the laser. 4. The super-resolution imaging system according to claim 1, wherein the first laser and the second laser are triggered synchronously, and the interval between the peaks of the two laser pulses is kept at 160ps-200ps. 5. The super-resolution imaging system according to claim 4, wherein the interval is 180 ps. 6. The super-resolution imaging system according to any one of claims 1-5, wherein the spatial light modulator is simultaneously loaded with a spiral gray-scale phase map for generating ring light and a coherent self-adaptive Grayscale phase map with optical aberration correction. 7. The super-resolution imaging system according to any one of claims 1-5, wherein the first pulse beam splitter divides the laser light into two paths according to 9:1, a small part of the light is transmitted into the spectrometer, and most of the light is transmitted into the spectrometer. The light is reflected as depleted light. 8. The super-resolution imaging system according to any one of claims 1-5, wherein the second pulse beam splitter divides the fluorescence into two parts according to 9:1, and a part of the light is reflected into the adaptive optical aberration A correction system for real-time correction of system aberrations; another part of the light is transmitted.