CN109358030B - Multicolor super-resolution microscope system with automatic alignment function - Google Patents
Multicolor super-resolution microscope system with automatic alignment function Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6445—Measuring fluorescence polarisation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses a multicolor super-resolution microscope system with an automatic alignment function, which belongs to the field of microscopic imaging. The system can avoid the influence of the beam drift on the performance of the super-resolution microscope system, simplify the system structure and increase the system stability. Meanwhile, compared with the prior method, the system disclosed by the invention can conveniently realize multicolor imaging.
Description
Technical Field
The invention relates to the field of microscopic imaging, in particular to a multicolor super-resolution microscope system with an automatic alignment function.
Background
Like most optical imaging, the abbe diffraction limit has been a constraint on the resolution of microscopic systems since the invention of microscopes. Early microscopy systems were all wide field imaging systems with limited imaging resolution. This was not improved until the invention of Confocal microscopy (Confocal microscopy). The basic concept of confocal Microscopy was proposed by Marvin Minsky in 1957 (see Microcopy Apparatus, U.S. Pat. No. 5, 3013467A), but the technique was not really instrumented until 1978 (see Christoph Cremer et al, Considerations on a laser-scanning-microscope with high resolution and depth field, Microcopia Acta 81, 31-44 (1978)). Compared with the traditional wide-field microscope system, the confocal microscope system adopts a scanning imaging mode, and a Pinhole (pinpole) is arranged on a focal plane conjugated with an imaging object plane to shield stray light around a non-imaging point, so that the effective point spread function of the system is effectively limited. The optical transfer function analysis of the system can prove that the limit resolution of the system can be improved by about 1.4 times by using a confocal method.
In recent years, with the proposal of Stimulated Emission Depletion microscopy (STED microscopy) (see steel w. hel et al Breaking the Diffraction resolution limit by simulation-Emission-Depletion fluorescence microscopy, Optics Letters 19, 780-782, 1994), the resolution of far-field optical microscopy is improved even more, and the resolution is further advanced to the nanometer level, allowing the observation of proteins on the nanometer scale on living cells, thus physically Breaking the Diffraction optical limit. The specific principle is as follows: based on the traditional confocal microscopy technology, fluorescence excitation is carried out on the fluorescence mark, meanwhile, another high-intensity coaxial laser beam is used for forming a hollow focusing dark spot, diffraction dispersion luminescence around the fluorescence mark is inhibited, and therefore only the fluorescence excitation phenomenon of the central point can be observed, the diffraction limit is broken, and the purpose of super-resolution microscopic imaging is achieved.
Although the STED microscopy technology effectively improves the imaging resolution of the optical fluorescence microscopy system, due to the limitation of the theory itself, one beam of suppression light beam needs to be added on the basis of the original excitation light, and the two beams of laser light are required to keep perfect alignment in the whole imaging process, so as to ensure the optimal image display effect. However, due to the high heat generated during the transmission of the laser, the deformation of the system device and the non-uniform refractive index of air caused by the temperature and humidity change in the operating environment are easily caused, and the like, the laser beam is prone to have problems of energy drift, parallel offset, angular offset, beam collimation degradation and the like, and the phenomenon becomes more serious with the passage of time, greatly affecting the precision of the microscope, and even affecting the service life of the device. Therefore, ensuring alignment of the excitation light and the suppression light has been a difficulty in STED microscopes.
In order to solve the above problems, one solution is to monitor the laser beam and adjust and compensate the generated offset in real time during the use of the device. Therefore, researchers have performed a lot of work for this purpose, such as the zuo and the wei (see the beam drift amount fast feedback control type high-precision laser collimation method and device, chinese patent ZL200410033610.8), the kui (see the beam drift real-time automatic correction and compensation method and device, chinese patent ZL 201110338933.8), and so on, which have proposed designs with similar functions. However, the above methods all require additional components to be added to the original microscope architecture for beam monitoring and drift compensation, thereby increasing system complexity and cost.
A more direct solution is to couple the excitation light and the suppression light into the same fiber, thereby achieving automatic alignment of the two lasers. However, since suppression of light generally requires additional phase modulation to optimize the corresponding point spread function, how to avoid the effect of phase modulation on the excitation light becomes a bottleneck for using this scheme. The above problems are solved to some extent by the wave plate based easystead design proposed in 2010 by Matthias Reuss et al (see Matthias Reuss et al, Optics Express 18, 1049-. However, since the wave plate itself is not an optical device having wavelength selective characteristics, in order to minimize the influence on the excitation light, the wavelength of the excitation light can be limited only to a narrow range according to the dispersion curve of the wave plate. Thus, the easySTED architecture is only suitable for monochromatic super-resolution microscopy imaging.
Disclosure of Invention
The invention aims to provide a multicolor super-resolution microscope system with an automatic alignment function, which has the multicolor imaging function while ensuring the automatic alignment function of an STED microscope, does not add additional devices and reduces the complexity of the system.
In order to achieve the above object, the multi-color super-resolution microscope system with an automatic alignment function provided by the invention comprises an illumination unit and a detection unit, wherein the illumination unit comprises an excitation light source and a suppression light source, an excitation light beam emitted by the excitation light source and a suppression light beam emitted by the suppression light source are combined into a beam of mixed light through a beam combining element, and a single-mode polarization maintaining optical fiber, a phase modulation component for modulating the phase of the suppression light beam, a first dichroic mirror, a first telescopic component, a scanning component, a second telescopic component, a microscope objective and a sample stage are sequentially arranged along the light path of the mixed light; the optical path of the suppression light beam is provided with a relative phase delay component which splits the suppression light beam into two polarization components which are vertical to each other and generates phase delay to the two polarization components so as to destroy the coherence between the two components; the detection unit comprises an imaging component arranged on a reflection light path of the first dichroic mirror, the imaging component is in communication connection with the computer and comprises at least one imaging module; the sample is excited by the mixed light to generate a fluorescent signal, the fluorescent signal returns to the first dichroic mirror along the original path and is reflected to the imaging component, the imaging component converts the optical signal into an electric signal and transmits the electric signal to the computer, and the computer reads the electric signal and restores the electric signal into a fluorescent image.
In the above technical solution, the suppression light beam from the suppression light source is output from the relative phase delay element and reaches the beam combining element, and is combined with the excitation light beam from the excitation light laser light source into a mixed light beam. The mixed light beam is coupled into the single-mode polarization maintaining fiber, and the mixed light beam emitted after passing through the single-mode polarization maintaining fiber sequentially passes through the phase modulation assembly, the first telescopic assembly, the scanning assembly and the second telescopic assembly, is incident into the microscope and then is focused into a sample placed on the sample stage. The fluorescence from the sample is reversely collected by the microscope objective, returns along the original path, is finally reflected by the dichroic mirror to enter the imaging assembly, and forms an observation image after being processed by a computer.
The beam combining element has the function of combining the excitation beam and the suppression beam into a mixed beam, and has various implementation schemes; preferably, the beam combining element selects a dichroic mirror that reflects the excitation light beam wavelength and transmits the suppression light beam wavelength.
Preferably, a first optical modulator for selecting a wavelength of the excitation light beam and modulating a transmission light intensity of the corresponding wavelength is disposed on an optical path of the excitation light beam, and a second optical modulator for modulating a transmission light intensity of the suppression light beam is disposed on an optical path of the suppression light beam. The first light modulator is preferably an acousto-optic tunable filter, and the wavelength of the excitation light required is determined by the fluorescent dye used in the sample, and preferred wavelengths include 485nm, 590nm and 650 nm. The second light modulator is preferably an acousto-optic modulator.
Preferably, the relative phase retardation assembly includes a first polarization beam splitter that splits the suppression light beam into a horizontal polarization component and a vertical polarization component, a first mirror for changing a direction of the horizontal polarization component, a second mirror for changing a direction of the vertical polarization component, and a second polarization beam splitter that combines the horizontal polarization component and the vertical polarization component.
In order to make the polarization direction of the horizontal polarization component parallel to the fast axis direction of the single-mode polarization-maintaining fiber, the relative phase retardation module preferably further comprises an 1/2 wave plate for rotating the polarization direction of the horizontal polarization component of the suppression light beam.
Preferably, the phase modulation component comprises a fourth reflector, a spatial light modulator, a second 1/4 wave plate, a third lens and a fifth reflector which are arranged in sequence along the optical path; the third lens is located at the middle position of the spatial light modulator and the fifth reflector, and the focal length of the third lens is equal to the distance between the third lens and the fifth reflector.
The mixed light beam firstly reaches a first phase diagram area of the spatial light modulator, and primary phase modulation is carried out on horizontal polarization components of the light suppression part in the mixed light beam; then the mixed light beam sequentially passes through a second 1/4 wave plate and a third lens, then returns after being reflected by a fifth reflector, passes through the third lens and a second 1/4 wave plate, and reaches a second phase image area of the spatial light modulator again for secondary phase modulation; the secondary phase modulation aims at inhibiting the vertical polarization component of the light part in the mixed light beam; after the modulated inhibiting light is focused by the microscope objective, the light intensity in the focusing light spot is distributed in a hollow way around the focus.
The spatial light modulator only has a phase modulation effect on linearly polarized light in a single direction, the corresponding working wavelength range is 750-850 nm, other visible light bands outside the corresponding working wavelength range only have a high reflectivity effect, and the spatial light modulator is preferably an X10468-02 type spatial light modulator of Hamamatsu corporation of Japan. The first phase diagram and the second phase diagram of the spatial light modulator are circular and are positioned in the horizontal central position of the effective area of the whole spatial light modulator, the positions of the two phase diagrams can be exchanged, and the radius of the two phase diagrams is equal to the effective aperture of the phase modulation component.
The scanning assembly is used for changing the deflection angle of the mixed light beam at the entrance pupil of the microscope objective so that the focus of the mixed light beam can scan two-dimensionally on the focal plane of the microscope objective in the sample. The first scanning mirror and the scanning component have equal effective aperture, and the scanning direction is vertical to the second scanning mirror and the third scanning mirror. The first scan mirror scans at a faster frequency than the second and third scan mirrors. Preferably, the scanning frequencies of the first scanning mirror, the second scanning mirror and the third scanning mirror are respectively 15KHz, 0.1KHz and 0.1 KHz.
Preferably, the first and second telescopic assemblies each comprise two convex lenses with convex surfaces facing away from each other and being confocal. The device is used for beam expanding (or beam reducing) collimation and maintains the conjugate relation between the scanning assembly and the back focal plane of the microscope objective in the system, and the magnification of the device is equal to the size of the entrance pupil of the microscope objective divided by the effective aperture of the scanning assembly.
Preferably, the imaging module comprises a second dichroic mirror for separating the fluorescence signal detected by the current imaging module from the fluorescence signal of the subsequent imaging module, a filter for filtering stray light signals not belonging to the outside of the spectrum of the detection channel, a fourth lens for focusing the collected fluorescence onto the photon counter, and the photon counter for linearly converting the fluorescence signal into an electrical signal according to the number of collected fluorescence photons.
Preferably, the photon counter is an avalanche photodiode or photomultiplier tube.
The sample stage is used for bearing a sample and providing three-dimensional movement capability, and preferably, the two-dimensional radial movement range of the sample stage is more than 5mm, the axial movement range is more than 100 mu m, and the movement precision is less than 1 mu m.
Compared with the prior art, the invention has the beneficial effects that:
(1) the number of optical elements of the existing system is simplified, the structure is simple, and the system is stable;
(2) the realization difficulty is low, and the cost is low;
(3) multicolor fluorescence imaging capabilities can be achieved.
Drawings
FIG. 1 is a schematic diagram of a multi-color super-resolution microscope system with auto-alignment function according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a relative phase delay element according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a phase modulating assembly according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a spatial light modulator according to an embodiment of the present invention;
FIG. 5 is a schematic structural diagram of a scanning assembly according to an embodiment of the present invention;
fig. 6 is a schematic structural diagram of an imaging assembly in an embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to the following embodiments and accompanying drawings.
Examples
Referring to fig. 1 to 6, the auto-alignment multi-color super-resolution microscope system of the present embodiment includes:
the device comprises an excitation light source 1, a suppression light source 2, a first light modulator 3, a second light modulator 4, a relative phase delay assembly 5, a beam combination element 6, a first 1/2 wave plate 7, a first lens 8, a single-mode polarization maintaining optical fiber 9, a second lens 10, a second 1/2 wave plate 11, a phase modulation assembly 12, a first dichroic mirror 13, a first telescope assembly 14, a scanning assembly 15, a second telescope assembly 16, a first reflecting mirror 17, a first 1/4 wave plate 18, a microscope objective lens 19, a sample stage 20, an imaging assembly 21 and a computer 22. All optical elements and fluorescent samples are located on a coaxial optical path.
In this embodiment, the excitation light beam from the excitation light source 1 passes through the first optical modulator 3 and reaches the beam combining element 6. The suppression light beam from the suppression light source 2 passes through the second light modulator 4 and then reaches the relative phase delay component 5; the output from the relative phase delay unit 5 reaches the beam combining element 6. The excitation beam and the suppression beam are combined into a mixed beam by the beam combining element 6. The mixed light beam passes through a first 1/2 wave plate 7 and then is focused and coupled into a single-mode polarization maintaining fiber 9 through a first lens 8. The mixed light beam emitted after passing through the single-mode polarization maintaining fiber 9 is expanded and collimated again by the second lens 10, and the mixed light beam after being expanded and collimated reaches the phase modulation component 12 through the second 1/2 wave plate 11. The mixed light beam output from the phase modulation unit 12 is transmitted through the first dichroic mirror 13, and then passes through the first telescopic unit 14 to reach the scanning unit 15. The mixed light beam emitted from the scanning assembly 15 passes through the second telescope assembly 16, is then deflected by the first reflector 17, passes through the first quarter-wave plate 18, enters the microscope objective lens 19, and is finally focused into the sample 23 placed on the sample stage 20. The fluorescence from the sample 12 is collected reversely by the microscope objective lens 19, passes through the first 1/4 wave plate 18, the first reflector 17, the second telescope component 16, the scanning component 15 in sequence, the first telescope component 14, and finally the dichroic mirror 13, is reflected into the imaging component 21, and is processed by the computer 22 to form an observation image.
In this embodiment, the excitation light source 1 provides an excitation light beam for the microscope system, which may be a white light laser or a laser group composed of a plurality of solid lasers, and this embodiment is a white light laser; the wavelength range is 450-700 nm. The average power of the exciting light source 1 is not less than 0.5 mW/nm; the excitation beam is linearly polarized with a purity higher than 1000: 1.
In the embodiment, the inhibition light source 2 provides an inhibition light beam for the microscope system, the wavelength of the light beam is longer than that of the excitation light beam, and the wavelength range is 770-775 nm; the average power of the light source 2 is inhibited to be more than 3W/nm; the light beam is restrained to be linearly polarized light with the purity higher than 10000: 1.
The excitation light source 1 and the suppression light source 2 may be continuous light lasers or pulse lasers. In this embodiment, when the two lasers are pulsed lasers, the pulse frequency of the lasers is between 20 MHz and 200MHz and kept synchronous, preferably 80MHz, wherein the pulse width of the laser light source should be less than 100ps, and the pulse width of the suppression light source 2 should be between 500 ps and 1000 ps.
The first optical modulator 3 is used for selecting a wavelength of a required excitation light beam and modulating transmission light intensity of a corresponding wavelength, the first optical modulator of the embodiment is an acousto-optic tunable filter, the wavelength of the required excitation light is determined by fluorescent dye used in a sample, and the preferable wavelengths include 485nm, 590nm and 650 nm. The second optical modulator 4 is used to modulate the transmitted intensity of the suppressed light, and the second optical modulator of the present embodiment is an acousto-optic modulator.
The relative phase delay element 5 is used to generate a phase delay for the two vertically polarized components of the input suppression beam that is large enough to break the coherence between the two components. There are various implementations of the phase delay component 5. The relative phase retardation assembly of the present embodiment includes a third 1/2 wave plate 24, a first polarizing beam splitter 25, a second polarizing beam splitter 26, a first mirror 27, a second mirror 28, and a glass block 29. The suppressed beam path into the opposing phase delay element 5 is shown by the arrows in fig. 2: the suppressed beam first passes through the third 1/2 wave plate 24 to reach the first polarizing beam splitter 25. The third 1/2 wave plate 24 is used to rotate the polarization direction of the rejection light beam, thereby adjusting the energy distribution of the rejection light beam after passing through the first polarization beam splitter 25. The suppression light beam is divided into a horizontal polarization component and a vertical polarization component after reaching the first polarization beam splitter 25, wherein the horizontal polarization component directly passes through the first polarization beam splitter 25 and then reaches the second polarization beam splitter 26 after being reflected by the first reflecting mirror 27; the vertically polarized light component is reflected by the first polarizing beam splitter 25, transmitted through the glass block 29, and reaches the second polarizing beam splitter 26. The glass block 29 is a BK-7 rectangular glass block, the long side of the rectangle is vertical to the incidence direction of the polarized light beam in the vertical direction, and the thickness of the glass block in the light transmission direction is more than 18 mm; the horizontally polarized component and the vertically polarized component are combined by the second polarization beam splitter 26 and then exit.
The suppressed beam emitted through the relative phase delay element 5 and the excitation beam emitted through the first optical modulator 3 are combined into a mixed beam by the beam combining element 6. There are various implementations of the beam combining element 6, and the beam combining element 6 of this embodiment selects a dichroic mirror that reflects the wavelength of the excitation light beam and transmits the wavelength of the suppression light beam.
The mixed light beam is emitted from the beam combining element 6, passes through the first 1/2 wave plate 7 and the first lens 8 in sequence, and is coupled into the single-mode polarization-maintaining fiber 9. The first 1/2 wave plate 7 is used for rotationally suppressing the horizontal polarization component of the light beam to make the polarization direction of the horizontal polarization component parallel to the fast axis direction of the single-mode polarization-maintaining fiber 9; the first lens 8 is a double cemented lens selected to reduce chromatic aberration, and the focal length of the first lens 8 is determined by the numerical aperture of the single-mode polarization maintaining fiber 9 and the radius of the mixed beam, and is expressed by the following formula:
f2=r2/NA
wherein f is1Is the focal length of the first lens 8, NA is the numerical aperture of the single-mode polarization-maintaining fiber 9, r1Is the mixed beam radius; length of single mode polarization maintaining fiber 9<1m。
The mixed light beam emitted from the single-mode polarization-maintaining light beam 9 is restored into a collimated light beam after passing through the second lens 10. Wherein, the second lens 10 is a double cemented lens selected to reduce chromatic aberration, and the focal length of the second lens 10 is determined by the numerical aperture of the single-mode polarization maintaining fiber 9 and the effective aperture of the phase modulation component 12, and is expressed by the following formula:
f2=r2/NA
wherein f is2Is the focal length of the second lens 10, and NA is the numerical aperture of the single-mode polarization-maintaining fiber 9, r2Is the effective aperture of the phase modulating component. The collimated mixed beam further passes through a second 1/2 wave plate 11. The second 1/2 wave plate 11 is used to rotate the original horizontal polarization component of the suppressed light beam in the emergent mixed light beam back to the horizontal position.
The mixed light beam exiting from the second 1/2 wave plate 11 enters the phase modulation assembly 12. The phase modulation component 12 is used to modulate the phase of the input suppressed light, and there are various implementations. The phase modulation assembly 12 of the present embodiment includes a fourth mirror 30, a spatial light modulator 31, a second 1/4 wave plate 32, a third lens 33, and a fifth mirror 34. The direction of transmission of the mixed beam within the phase modulating assembly 12 is shown by the arrows in fig. 3: the mixed light beam firstly passes through a fourth reflector 30 to reach a first phase diagram area of a spatial light modulator 31 for the first time, and primary phase modulation is carried out on horizontal polarization components of the light suppression part in the mixed light beam; then, the mixed light beam sequentially passes through the second 1/4 wave plate 32 and the third lens 33, is reflected by the fifth reflector 34 and then returns, passes through the third lens 33 and the second 1/4 wave plate 32, and finally reaches the second phase diagram region of the spatial light modulator 31 for secondary phase modulation; the secondary phase modulation aims at inhibiting the vertical polarization component of the light part in the mixed light beam; after the modulated inhibiting light is focused by the microscope objective, the light intensity in the focusing light spot is distributed in a hollow way around the focus.
The spatial light modulator 31 only has a phase modulation effect on linearly polarized light in a single direction, the corresponding working wavelength range is 750-850 nm, the other visible light bands outside the corresponding working wavelength range only show a high reflectivity effect, and an X10468-02 type spatial light modulator of Hamamatsu corporation of Japan is selected. The first and second phase patterns of the spatial light modulator 31 are circular and are horizontally centered (as shown in fig. 4) over the active area of the spatial light modulator, and the positions of the two phase patterns can be interchanged, with a radius as large as the effective aperture of the phase modulating element. The third lens 33 is located at a position right in the middle between the spatial light modulator 31 and the fifth mirror 34, and the focal length of the lens is equal to the distance between the third lens 33 and the fifth mirror 34. After the modulated inhibiting light is focused by the microscope objective lens 19, the light intensity in the focused light spot is distributed in a hollow way around the focus.
The mixed light beam emitted from the phase modulation element 12 passes through the first dichroic mirror 13, and then reaches the scanning element 15 through the first telescopic element 14. Wherein the first dichroic mirror has no effect on the propagation of the mixed light beam. The first telescope assembly 14 includes two convex, back-to-back convex lenses, which are confocal and used to expand (or reduce) the beam, collimate and maintain the conjugate relationship between the phase modulating assembly 12 and the scanning assembly 15 in the system, and the magnification is equal to the effective aperture of the scanning assembly 12 divided by the effective aperture of the phase modulating assembly 15.
The scanning assembly 15 is used for changing the deflection angle of the mixed light beam at the entrance pupil of the microscope objective 19 to enable the focus of the mixed light beam to scan on the focal plane of the microscope objective in the sample in two dimensions, and various implementation schemes are provided. The scanning assembly 15 of the present embodiment includes a first scanning mirror 35, a second scanning mirror 36 and a third scanning mirror 37, and the propagation path of the mixed light beam in the scanning assembly 15 is shown by the arrows in fig. 5. The first scan mirror 35 has an effective aperture as large as the effective aperture of the scan element 15, and the scanning direction is perpendicular to the second scan mirror 36 and the third scan mirror 37. The first scan mirror 35 scans at a faster frequency than the second and third scan mirrors 36, 37. The scanning frequencies of the first, second and third scan mirrors 35, 36, 37 are 15KHz, 0.1KHz and 0.1KHz, respectively.
The mixed light beam emitted from the scanning assembly 15 passes through the second telescope assembly 16, is reflected by the reflector 17, then passes through the first 1/4 wave plate 18, enters the entrance pupil of the microscope objective lens 19, and is finally focused in the sample 23 placed on the sample stage 20.
The second telescope assembly 16 includes two convex lenses with convex surfaces facing back and being confocal, and is used for expanding beam (or reducing beam) and collimating and maintaining the conjugate relation between the scanning assembly 15 and the back focal plane of the microscope objective 19 in the system, and the magnification of the second telescope assembly is equal to the entrance pupil size of the microscope objective divided by the effective aperture of the scanning assembly.
The first 1/4 wave plate 18 functions to change the polarization of the mixed beam from linear to circular.
The microscope objective 19 is used for focusing the mixed light beam on the sample for illumination and reversely collecting fluorescence from the sample; in order to ensure the resolution, a flat field achromatic immersion type microscope objective with the numerical aperture larger than 1.05 and the magnification of 60-100 times is selected.
The sample stage 20 is used for bearing a sample 23 and providing three-dimensional movement capability; the two-dimensional radial movement range of the sample stage 20 is larger than 5mm, the axial movement range is larger than 100 μm, and the movement precision is smaller than 1 μm.
Under illumination by the mixed beam, the sample 23 fluoresces and is collected back by the microscope objective 19. The fluorescence sequentially passes through the first 1/4 wave plate 18, the first reflector 17, the second telescopic system 16, the scanning assembly 15 and the first telescopic system 14, and is finally reflected by the first dichroic mirror to enter the imaging assembly 21. The imaging assembly 21 may comprise 1 or more basic imaging modules according to whether multicolor imaging is required, as shown in fig. 6, the present embodiment comprises three basic imaging modules, wherein the first basic imaging module 38, the second basic imaging module 39 and the third basic imaging module 40 are all composed of the same optical elements, and the structures are also completely the same. Taking the first basic imaging module 38 as an example, the second dichroic mirror 41, the filter 42, the fourth lens 43 and the single photon counter 44 are included.
The third dichroic mirror 41 is used for separating the channel detection fluorescence signal from the subsequent channel fluorescence signal, and the transmission filter curve is determined by the detected fluorescence spectrum, wherein the high reflectivity (> 95%) of the channel fluorescence spectrum is maintained, and the high transmission filter (> 98%) of the subsequent channel detection fluorescence spectrum is maintained; the filter 42 functions to filter stray light signals that do not fall outside the detection channel spectrum; the filter 42 is formed by stacking 2 or more than 2 narrow-band filters having the same transmission and filtration curve. The fourth lens 43 functions to focus the collected fluorescence onto the photon counter, and the fourth lens 43 of the present embodiment selects a double cemented lens with a focal length of 100 mm. The single photon counter 44 is used for linearly converting the fluorescence signal into an electric signal according to the number of the collected fluorescence photons; the photon counter of the present embodiment is selected from an avalanche photodiode or a photomultiplier tube. The electrical signals generated by the single photon counter 44 are ultimately read by the computer 22 and reduced to a fluorescent image.
Claims (8)
1. A multi-color super-resolution microscope system with automatic alignment function comprises an illumination unit and a detection unit, and is characterized in that:
the illumination unit comprises an excitation light source and an inhibition light source, an excitation light beam emitted by the excitation light source and an inhibition light beam emitted by the inhibition light source are combined into a beam of mixed light through a beam combining element, and a single-mode polarization maintaining optical fiber, a phase modulation component for modulating the phase of the inhibition light beam, a first dichroic mirror, a first telescopic component, a scanning component, a second telescopic component, a microscope objective and a sample stage are sequentially arranged along the light path of the mixed light;
the light path of the suppression light beam is provided with a relative phase delay component which splits the suppression light beam into two polarization components which are vertical to each other and generates phase delay for the two polarization components so as to destroy the coherence between the two components; the relative phase delay assembly comprises a first polarization beam splitter for splitting the suppression beam into a horizontal polarization component and a vertical polarization component, a first reflecting mirror for changing the direction of the horizontal polarization component, a second reflecting mirror for changing the direction of the vertical polarization component, a second polarization beam splitter for combining the horizontal polarization component and the vertical polarization component, and a 1/2 wave plate for rotating the polarization direction of the horizontal polarization component of the suppression beam;
the detection unit comprises an imaging component arranged on a reflection light path of the first dichroic mirror, and the imaging component is in communication connection with a computer and comprises at least one imaging module; the sample is excited by the mixed light to generate a fluorescent signal, returns to the first dichroic mirror along the original path and then is reflected to the imaging component, the imaging component converts the optical signal into an electric signal and transmits the electric signal to the computer, and the computer reads the electric signal and restores the electric signal into a fluorescent image.
2. The multi-color super-resolution microscope system according to claim 1, wherein: the light path of the exciting light beam is provided with a first light modulator used for selecting the wavelength of the exciting light beam and modulating the transmission light intensity of the corresponding wavelength, and the light path of the inhibiting light beam is provided with a second light modulator used for modulating the transmission light intensity of the inhibiting light beam.
3. The multi-color super-resolution microscope system according to claim 1, wherein: the phase modulation component comprises a fourth reflector, a spatial light modulator, a second 1/4 wave plate, a third lens and a fifth reflector which are sequentially arranged along a light path;
the third lens is located in the middle of the spatial light modulator and the fifth reflector, and the focal length of the third lens is equal to the distance between the third lens and the fifth reflector.
4. The multi-color super-resolution microscope system according to claim 1, wherein: the scanning component comprises a first scanning mirror, a second scanning mirror and a third scanning mirror which are sequentially arranged along a light path, and the scanning direction of the first scanning mirror is vertical to the scanning direction of the second scanning mirror and the scanning direction of the third scanning mirror.
5. The multi-color super-resolution microscope system according to claim 1, wherein: the first telescope component and the second telescope component both comprise two convex lenses with convex surfaces arranged backwards and confocal.
6. The multi-color super-resolution microscope system according to claim 1, wherein: the imaging module comprises a second dichroic mirror used for separating a fluorescence signal detected by the current imaging module from a fluorescence signal of a subsequent imaging module, an optical filter used for filtering a stray light signal which does not belong to the outside of a detection channel spectrum, a fourth lens used for focusing the collected fluorescence on the photon counter, and the photon counter used for linearly converting the fluorescence signal into an electric signal according to the number of the collected fluorescence photons.
7. The multi-color super-resolution microscope system according to claim 6, wherein: the photon counter is an avalanche type light emitting diode or a photomultiplier.
8. The multi-color super-resolution microscope system according to claim 1, wherein: the two-dimensional radial movement range of the sample stage is larger than 5mm, the axial movement range is larger than 100 mu m, and the movement precision is smaller than 1 mu m.
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| CN113466187B (en) * | 2021-04-12 | 2022-11-08 | 清华大学 | System and method for carrying out polarization super-resolution imaging on fluorescence anisotropy |
| CN113325563B (en) * | 2021-04-21 | 2022-06-10 | 浙江大学 | Multicolor three-dimensional super-resolution expansion microscope system with large view field |
| CN114019765B (en) * | 2021-10-25 | 2023-09-19 | 之江实验室 | Common-path phase modulation laser direct writing method and device based on edge light suppression |
| CN114967089B (en) * | 2022-05-17 | 2023-04-07 | 浙江大学 | Three-dimensional super-resolution microscopic imaging system with automatic beam collimation |
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