CN107941777B - Anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system - Google Patents
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Abstract
The invention discloses an anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system which comprises a first laser, a beam expanding and collimating unit, a phase grating, a third lens, a first dichroic mirror, an objective lens, a sample stage, an optical filter, a fourth lens, a detector, a second dichroic mirror and a second laser. The invention utilizes the time-domain focusing characteristic to only generate the fluorescence excitation of the sample on the time-domain focusing plane and simultaneously realize the excitation of the fluorescence in the whole time-domain focusing plane. Therefore, compared with the prior art, the method can avoid bleaching the sample in the non-focusing plane and effectively improve the working efficiency of the SMS fluorescence microscopy technology.
Description
Technical Field
The invention relates to the field of optical instruments and biomedical microscopic imaging, in particular to an anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system and method.
Background
At present, the requirement of research in the field of biological medicine on the resolution of a microscope system is higher and higher, researchers need to know the three-dimensional structures of various substances in micro forms, however, the spot size of the traditional white-light wide-field microscope and the traditional laser confocal microscope cannot achieve the resolution, and the super-resolution microscope system perfectly solves the problem. In super-resolution microscopy, the current research focus is mostly on various fluorescence microscopy technologies, such as stimulated emission depletion (STED) fluorescence microscopy and single molecule localization fluorescence microscopy (SMS). Compared with the STED fluorescence microscopy, the SMS fluorescence microscopy has simpler optical path design and mechanical structure, does not need the combination of multiple beams, and is widely used in practice. The basic principle of the SMS fluorescence microscopy is that the protein is marked by PA-GFP, the energy of a 405nm laser is adjusted, the cell surface is irradiated by low energy, only a few fluorescent molecules which are sparsely distributed in a visual field are activated at a time, then the fluorescent single molecules are accurately positioned by illumination of 488nm laser, and the fluorescent single molecules are detected by Gaussian fitting. After the position of these molecules is determined, 488nm laser irradiation is used for a long time to bleach the correctly positioned fluorescent molecules, so that they can not be reactivated by the laser of the next round. Thereafter, other fluorescent molecules are activated and bleached with 405nm and 488nm lasers, respectively, into the next cycle. After this cycle has been continued over ten thousand times, we will get an accurate localization of all fluorescent molecules inside the cell.
Betzig et al, in journal Science, 313, mention the use of broad field illumination and continuous laser as a light source to simplify the construction of SMS fluorescence microscopy systems. However, if three-dimensional imaging of a large depth (imaging range greater than 1 μm in the depth direction) is to be achieved for a sample, it is necessary to change the height of the sample in conjunction with the micro-displacement stage. In this mode of operation, the entire sample, including the fluorescent proteins in the non-viewing plane regions, will be excited throughout the imaging process. The non-observed planar fluorescent protein is excited for a long time, so that the fluorescent protein loses activity and is bleached. When the micro-displacement platform continues to move, the original non-observation area becomes an imaging plane, and the imaging plane cannot realize the process of re-excitation and imaging of the fluorescent protein because the fluorescent protein is bleached before. This problem is particularly evident when deep tissue imaging is performed using SMS fluorescence microscopy. Therefore, the existing three-dimensional SMS does not well meet the actual demand.
Disclosure of Invention
The invention provides a novel anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system, aiming at the problems that fluorescent protein molecules are bleached and the like when the three-dimensional SMS fluorescence microscopic technology is used for scanning in the depth direction. The system utilizes the time domain focusing characteristic, does not bleach the fluorescent protein molecules of a non-focusing plane, has simple structure and good universality, and can also be used for two-dimensional imaging.
An anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system comprises a first laser, a beam expanding and collimating unit, a phase grating, a third lens, a first dichroic mirror, an objective lens, a sample stage capable of moving to and fro opposite to the objective lens, an optical filter, a fourth lens, a detector, a second dichroic mirror and a second laser;
laser emitted by the first laser is subjected to beam expanding and collimating by the beam expanding and collimating unit, obliquely enters the surface of the phase grating, is reflected by the phase grating and then irradiates the second dichroic mirror;
after being reflected by a second dichroic mirror, the laser emitted by the second laser is combined with the laser from the first laser and transmitted by a second dichroic mirror, and the laser is focused on the back focal plane of an objective lens after passing through a third lens and the first dichroic mirror, so that the whole sample on the sample stage is illuminated in a wide field, and a part of a fluorescent mark in the sample is activated;
after the laser emitted by the first laser is emitted from the objective lens, time-domain focusing is generated at a focal plane of the objective lens, and fluorescence of a sample which is positioned on the sample stage and is activated by the second laser is excited;
when the sample stage moves, the time domain focusing plane of the laser emitted by the objective lens is positioned at different depths of the sample, so that the fluorescence at different depths of the sample is excited, and the activated fluorescence mark in the sample is scanned layer by layer;
the excited fluorescence in the sample is transmitted along the direction opposite to the laser propagation direction, reenters the objective lens, is reflected by the first dichroic mirror, then sequentially passes through the optical filter and the fourth lens, and finally is received and imaged by the detector.
The multiple fluorescence images obtained by the detector of the invention generally need to be spliced by a signal processing unit to the fluorescence signals of each time domain focusing plane received by the detector, so as to obtain the three-dimensional super-resolution structure of the whole sample. The signal processing unit can be a single splicing image processor specially matched with the three-dimensional super-resolution microscope system, and can also directly adopt the existing industrial computer. Certainly, for convenience of research and analysis, the three-dimensional super-resolution microscope system of the present invention may further include a display having an image display function, or/and a parameter setting function, or/and a command input function, or may be a display specially configured for the three-dimensional super-resolution microscope system of the present invention, or may directly employ a display or the like configured with an existing industrial computer. The splicing operation is prior art and can be implemented by existing software or programming.
Preferably, the beam expanding and collimating unit includes a first lens and a second lens coaxially disposed, the first lens and the second lens are disposed in a confocal manner, the first lens is disposed close to the first laser, and a focal length of the first lens is smaller than that of the second lens. In the invention, the first lens and the second lens are used for expanding and collimating the laser beam, so that the diameter of the laser beam is increased, the divergence angle is reduced, the laser beam is closer to parallel light, and the laser beam is converged to form smaller light spots; the first lens and the second lens are generally convex lenses.
In the present invention, the time-domain focusing refers to a focusing effect in which all frequencies of a single femtosecond pulse are only heavily summed on a focusing plane of an objective lens and are in phase with each other, thereby making the width of the pulse extremely narrow in the time domain; i.e. time-domain focusing, the whole focal plane will be focused, and the focal depth is extremely small, only exciting the fluorescence of the extremely thin layer of the sample in the time-domain focal plane.
The once working cycle of the anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system comprises the following steps:
step A: after being reflected by a second dichroic mirror, the laser emitted by the second laser is combined with the laser from the first laser, enters an objective lens after passing through a third lens and a first dichroic mirror, illuminates the whole sample on the sample stage in a wide field, and activates the fluorescent mark of part in the sample;
and B: laser emitted by the first laser passes through the first lens and the second lens, is obliquely incident on the surface of the phase grating and is reflected by the phase grating; the laser reflected by the phase grating passes through a second dichroic mirror, a third lens and a first dichroic mirror and is focused on the back focal plane of the objective lens; laser emitted by the first laser emits from the objective lens and then generates time-domain focusing at a focal plane of the objective lens, and fluorescence of a sample which is positioned on the sample stage and is activated by the second laser and positioned on the time-domain focusing plane is excited; in combination with the movement of the sample stage relative to the objective lens, the time-domain focusing plane of the laser emitted by the objective lens can be positioned at different depths of the sample, so that the fluorescence at different depths of the sample is excited, and the activated fluorescence mark in the sample in the step A is scanned layer by layer; the excited fluorescence in the sample is transmitted along the direction opposite to the laser propagation direction, reenters the objective lens and is reflected by the first dichroic mirror; the reflected fluorescence sequentially passes through the optical filter and the fourth lens, and is received and imaged by the detector;
and C: the laser emitted by the second laser continuously irradiates the sample until the fluorescent mark of part of the sample activated in the step A is bleached;
repeating the duty cycle of step A, B, C until all molecules in the sample are mapped; and finally, splicing the fluorescence signals of each time domain focusing plane received by the detector through a signal processing unit to obtain the three-dimensional super-resolution structure of the whole sample.
By controlling the intensity of the second laser's emitted light (less than 1 milliwatt), the proportion of the fluorescent labels that are reactivated (< 1%) is controlled.
Preferably, in the present invention, the first laser is a femtosecond laser, the intensity of the emitted laser is high, the pulse width is in the order of femtosecond, and the spectral bandwidth of the emitted laser is 10 nm.
More preferably, the first laser is a titanium sapphire femtosecond laser, the final output peak wavelength is 830 nm, the spectral bandwidth is 10 nm, the pulse width is less than 100 fs, and the output average power is 1.5 w. By adopting the technical scheme, the bleaching of the fluorescence mark of the non-imaging plane can be reduced while the two-photon excitation efficiency is ensured, and the imaging quality of the system in the whole three-dimensional imaging area is improved.
Preferably, in the present invention, the second laser is a continuous laser. More preferably, the second laser is a continuous optical semiconductor laser and has an output wavelength of 405 nm.
In the invention, the phase grating is used for adjusting the phase of the laser emitted by the first laser, so that diffraction angles of the same diffraction orders of the laser beams with different wavelengths are different, namely the laser is subjected to dispersion, the reflection angles of the laser beams with different wavelengths in the laser beams on the surface of the phase grating are different, but the laser beams with the same wavelength are still parallel light. According to the invention, by introducing the phase grating, the paths of the light with different wavelengths in the laser emitted by the first laser in the optical element are different, and the pulse width of the femtosecond laser is widened finally. More preferably, the expanded and collimated laser light is incident on the surface of the phase grating at 26.44 °.
In the invention, the third lens is used for focusing the laser reflected by the phase grating, and the focusing position is located at the back focal plane of the objective lens, wherein the laser with different frequencies in the laser beam is converged at different positions of the back focal plane of the objective lens.
In the present invention, the second dichroic mirror is configured to combine light beams from the first laser and the second laser. Preferably, the second dichroic mirror has high reflectivity of more than 90% for 405nm laser light and high transmittance of more than 90% for 825 to 835 nm laser light.
In the invention, the first dichroic mirror is used for transmitting laser and reflecting fluorescence of a sample. Preferably, the first dichroic mirror is placed at an angle of 45 ° with respect to the third lens. Preferably, the first dichroic mirror comprises two transmission bands: 825 nm to 835 nm and 400 to 410 nm, respectively; meanwhile, the first dichroic mirror reflects light from 500 nm to 600 nm, and the reflectivity is greater than 95%.
In the invention, the optical filter only transmits the fluorescence of the sample and filters the rest stray light.
Preferably, the fourth lens is a field lens, which can effectively reduce the size of the detector.
The terms "first", "second", "third", and the like, as used herein, are used only for distinguishing between different elements, and unless otherwise specified, do not limit the order, structure, and function of connecting the elements.
The invention utilizes the time-domain focusing characteristic to only generate the fluorescence excitation of the sample on the time-domain focusing plane and simultaneously realize the excitation of the fluorescence in the whole time-domain focusing plane. Compared with the prior art, the invention has the beneficial effects that:
1. the invention can effectively overcome the problem that the traditional SMS fluorescence microscopy can excite the fluorescence of non-observation areas near a focal plane, even near several micrometers of the focal plane, and the like, and avoid bleaching.
2. The invention can realize the excitation of fluorescence in the whole focusing plane and can effectively improve the working efficiency of the SMS fluorescence microscopy.
Drawings
FIG. 1 is an optical path diagram of an embodiment of an anti-bleaching single-molecule localization three-dimensional super-resolution microscopy system of the present invention;
wherein: 1. a first laser; 2. a first lens; 3. a second lens; 4. a phase grating; 5. a third lens; 6. a first dichroic mirror; 7. an objective lens; 8. a sample stage; 9. an optical filter; 10. a fourth lens; 11. a detector; 12. a second dichroic mirror; 13. a second laser.
Detailed Description
The present invention will be described with reference to the accompanying drawings, but the present invention is not limited thereto.
FIG. 1 is a light path diagram of an embodiment of an anti-bleaching monomolecular positioning three-dimensional super-resolution microscope system according to the present invention. The super-resolution microscope system of this embodiment includes:
1. a first laser; 2. a first lens; 3. a second lens; 4. a phase grating; 5. a third lens; 6. a first dichroic mirror; 7. an objective lens; 8. a sample stage; 9. an optical filter; 10. a fourth lens; 11. a detector; 12. a second dichroic mirror; 13. a second laser;
among them, the first laser 1 is preferably a Mai Tai femtosecond laser from Spectra-Physics. The wave band emitted by the first laser 1 is from 825 nanometers to 835 nanometers, the peak wavelength is 830 nanometers, the pulse width is less than 100 femtoseconds, and the laser with the power of 1.5 watts passes through the first lens 2 and the second lens 3 and then is expanded and collimated. The laser after beam expansion and collimation is incident on the surface of the phase grating 4 at 26.44 degrees to generate dispersion, that is, in a waveband from 825 nanometers to 835 nanometers in the laser beam, light with different wavelengths generates different diffraction angles. The longer the wavelength of light in the 825 nm to 835 nm band, the larger the diffraction angle.
The phase grating 4 is preferably a GR25-1208 type reflection line diffraction grating manufactured by THORLABS corporation; the focal length of the first lens 2 is 50 nanometers, the focal length of the second lens 3 is 200 nanometers, and both the first lens 2 and the second lens 3 are convex lenses.
The second laser 13 is preferably a PowerLine continuous optical semiconductor laser of Coherent, and has a center wavelength of 405 nm. After the laser beam from the second laser 13 and the laser beam of the first laser 1 dispersed by the phase grating 4 are combined through the second dichroic mirror 12, the combined laser beam passes through the third lens 5 and the first dichroic mirror 6 and is focused at the back focal plane of the objective lens 7, the whole sample is illuminated in a wide field, and the bleached fluorescent mark in the sample is activated; by controlling the intensity of the second laser's emitted light (less than 1 milliwatt), the proportion of the fluorescent labels that are reactivated (< 1%) is controlled.
Wherein the second dichroic mirror 12 is placed at 45 ° tilt and has high reflectivity for 405nm laser light and high transmittance for 825 to 835 nm laser light, preferably a 750 dcxxx type dichroic mirror from Chroma corporation.
The third lens 5 is a convex lens, the spherical surface of which is arranged opposite to the laser beam after being combined, and the optical axis of the third lens 5 is parallel to the laser beam after being combined.
Wherein, first dichroic mirror 6 slope 45 places, and first dichroic mirror 6 includes two transmission bands: 825 nm to 835 nm and 400 to 410 nm, respectively, while first dichroic mirror 6 reflects light from 500 nm to 600 nm (reflectance > 95%).
The light beam from the first laser 1 is focused and emitted at the back focal plane of the objective lens 7, the light with the same wavelength in the light beam is still parallel, and the light with the wave band from 825 nanometers to 835 nanometers generates time-domain focusing at the focal plane of the objective lens 7. The sample on the sample stage 8 is calibrated by PA-GFP fluorescent protein in advance, and after the sample in the time domain focusing plane is irradiated, the fluorescence is excited, and the central wavelength of the fluorescence is 510 nanometers. The focal depth of time-domain focusing is determined by a system point spread function, a specific calculation formula can be obtained from Simultaneous spatial and temporal focusing of femtocell pulses of the 13 th edition of the publication Optics Express 2005, and the focal depth is 750 nm in the embodiment calculated by using the formula.
By moving the sample stage 8 up and down, the time-domain focusing plane of the laser emitted by the objective lens 7 can be positioned at different depths of the sample, so that the sample can be scanned layer by layer; the fluorescence of the sample is transmitted along the direction opposite to the laser propagation direction, reenters the objective lens 7 and is reflected by the first dichroic mirror 6; the reflected fluorescence passes through the filter 9 and the fourth lens 10 in this order, and is received by the detector 11 to be imaged.
Among them, the filter 9 is preferably an FF01-514/44-25 band-pass filter of Semrock, and a combination of a plurality of FFs 01-514/44-25 band-pass filters, preferably 2 sheets, may be used in order to secure the filtering efficiency.
The fourth lens 10 is a convex lens.
Among them, the detector 11 is preferably an ORCA-Flash4.0C13440-20CU model S-CMOS camera manufactured by Hamamatsu corporation.
And the subsequent signal processing unit splices the fluorescence signals of the time domain focal plane of each layer received by the detector 11, that is: after the initial data (multiple fluorescence signals) are obtained, the stitching of the final three-dimensional image can be done by a variety of prior art techniques, such as Maliang algorithm developed by Huangchang et al (Tingwei Quan, Pengcheng Li, Fan Long, Shaoqun Zeng, Qingming Luo, Per Niklasheld, Gerd Ulrich Nienhaus, and Zhen-Li Huang, "Ultra-fast, high-precision image analysis for localization-based super resolution microscopy," Opt.express 18,11867-11876(2010)), to obtain the three-dimensional super-resolution structure of the entire sample. In this embodiment, the signal processing unit may be an industrial computer.
The invention utilizes the time-domain focusing characteristic to only generate the fluorescence excitation of the sample on the time-domain focusing plane and simultaneously realize the excitation of the fluorescence in the whole time-domain focusing plane. Therefore, compared with the prior art, the method can avoid bleaching the sample in the non-focusing plane and effectively improve the working efficiency of the SMS fluorescence microscopy technology.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. An anti-bleaching monomolecular positioning three-dimensional super-resolution microscopic system is characterized by comprising a first laser, a beam expanding collimation unit, a phase grating, a third lens, a first dichroic mirror, an objective lens, a sample stage capable of moving to and fro just opposite to the objective lens, an optical filter, a fourth lens, a detector, a second dichroic mirror and a second laser;
laser emitted by the first laser is subjected to beam expanding and collimating by the beam expanding and collimating unit, obliquely enters the surface of the phase grating, is reflected by the phase grating and then irradiates the second dichroic mirror;
after being reflected by a second dichroic mirror, the laser emitted by the second laser is combined with the laser from the first laser and transmitted by a second dichroic mirror, and then passes through a third lens and the first dichroic mirror and is focused on the back focal plane of the objective lens, and finally the whole sample on the sample stage is illuminated in a wide field, and the fluorescence mark of part of the sample is activated;
after the laser emitted by the first laser is emitted from the objective lens, time-domain focusing is generated at a focal plane of the objective lens, and fluorescence of a sample which is positioned on the sample stage and is activated by the second laser is excited;
the excited fluorescence in the sample is transmitted along the direction opposite to the laser propagation direction, reenters the objective lens, is reflected by the first dichroic mirror, then sequentially passes through the optical filter and the fourth lens, and is finally received and imaged by the detector;
the first laser is a femtosecond laser; the second laser is a continuous light laser.
2. The anti-bleaching monomolecular positioning three-dimensional super-resolution microscope system according to claim 1, wherein the beam expanding and collimating unit comprises a first lens and a second lens coaxially arranged, the first lens and the second lens are disposed in a confocal manner, the first lens is arranged close to the first laser, and the focal length of the first lens is smaller than that of the second lens.
3. The bleach resistant single molecule positioning three dimensional super resolution microscopy system as defined in claim 1 wherein said first laser is a titanium sapphire femtosecond laser with a final output peak wavelength of 830 nm, spectral bandwidth of 10 nm, pulse width of less than 100 fs and output average power of 1.5 w.
4. The bleach resistant single molecule positioning three dimensional super resolution microscopy system as defined in claim 1 wherein said second laser is a continuous optical semiconductor laser with an output wavelength of 405 nanometers.
5. The anti-bleaching monomolecular positioning three-dimensional super-resolution microscope system according to claim 1, wherein the expanded and collimated laser is incident on the surface of the phase grating at 26.44 °.
6. The anti-bleaching monomolecular positioning three-dimensional super-resolution microscopy system according to claim 4, wherein the second dichroic mirror has a high reflectivity for 405nm laser light and a high transmittance for 825 to 835 nm laser light.
7. The anti-bleaching monomolecular positioning three-dimensional super-resolution microscopy system according to claim 1, wherein the first and second dichroic mirrors are both tilted at 45 °.
8. The bleach resistant monomolecular positioning three-dimensional super-resolution microscopy system according to claim 6, wherein the first dichroic mirror comprises two transmission bands: 825 nm to 835 nm and 400 to 410 nm, respectively; meanwhile, the first dichroic mirror reflects light from 500 nm to 600 nm, and the reflectivity is greater than 95%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592278A (en) * | 2013-11-21 | 2014-02-19 | 中国计量学院 | Random positioning super-resolution microscopy method and device based on fluorescence-emission kill mechanism |
| CN104568874A (en) * | 2014-12-22 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Super-resolution microscope for imaging fluorescent substances |
| CN107389631A (en) * | 2017-04-28 | 2017-11-24 | 中国科学院生物物理研究所 | The multi-modal Structured Illumination super-resolution micro imaging system of high speed polychrome and its method |
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592278A (en) * | 2013-11-21 | 2014-02-19 | 中国计量学院 | Random positioning super-resolution microscopy method and device based on fluorescence-emission kill mechanism |
| CN104568874A (en) * | 2014-12-22 | 2015-04-29 | 中国科学院苏州生物医学工程技术研究所 | Super-resolution microscope for imaging fluorescent substances |
| CN107389631A (en) * | 2017-04-28 | 2017-11-24 | 中国科学院生物物理研究所 | The multi-modal Structured Illumination super-resolution micro imaging system of high speed polychrome and its method |
Non-Patent Citations (4)
| Title |
|---|
| 《Single-Molecule Super-Resolution Light-Sheet Microscopy》;Ying S. Hu et al;《ChemPhysChem》;20141231;第15卷;全文 * |
| Multilayer three-dimensional super resolution imaging of thick biological samples;Alipasha Vaziri et al;《PNAS》;20081223;第105卷(第51期);第20221右栏最后一段至第20225页 * |
| 可逆饱和光转移过程的荧光超分辨显微术;郝翔等;《激光与光电子学进展》;20121231;全文 * |
| 结构光照明荧光显微镜突破衍射极限的原理和在生命科学中的应用;吴美瑞等;《激光与光电子学进展》;20151231;全文 * |
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