CN111879737A - Device and method for generating high-flux super-diffraction limit focal spot - Google Patents
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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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/21—Polarisation-affecting properties
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- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/21—Polarisation-affecting properties
- G01N2021/216—Polarisation-affecting properties using circular polarised light
Abstract
The invention provides a method for generating high-flux super-diffraction-limit focal spots, which comprises the following steps: 1) generating exciting light; 2) modulating the generated loss light into hollow loss light; 3) combining the excitation light and the hollow loss beam and then converting the combined beam into a light beam array; 4) and focusing the light beam array on the sample to perform stimulated emission loss to generate a high-flux super-diffraction limit focal spot scanning sample. The invention also provides a device for generating the high-flux super-diffraction-limit focal spot. Compared with the prior art, the invention has the advantages that: the method has extremely high stimulated emission loss microscopic imaging speed and laser direct writing photoetching speed; with ultra-high resolution down to tens of nanometers.
Description
Technical Field
The invention belongs to the field of optical engineering, and particularly relates to a device and a method for generating high-flux super-diffraction limit focal spots.
Background
The resolution of far-field optical microscopes has been limited to essentially half the wavelength of the excitation light since the beginning of the 20 th century because of the diffraction limit. While electron and scanning probe microscopes provide finer detail, they do not provide non-invasive three-dimensional imaging of living cells and tissues and other transparent materials. Various far-field super-resolution microscopic imaging methods have been proposed later, and the stimulated emission depletion microscopy technology has been developed most mature and widely applied.
The stimulated emission depletion microscopy technology is mainly characterized in that a hollow loss light beam is covered on a diffraction-limited excitation light beam, so that a fluorophore excited at the outer ring of a facula instantaneously returns to a ground state, and a fluorescent molecule excited at the center of the facula normally emits fluorescence to be received as an effective signal, so that the resolution far beyond the diffraction limit is obtained. However, the stimulated emission depletion microscopy is used for imaging in a single-point scanning mode, long imaging time is needed, the microscopic imaging in a large field range is limited, and meanwhile, the light intensity of the lost light is high, so that the photobleaching of a sample is easily caused.
Meanwhile, the laser direct writing lithography technology acts on a sample material through a focusing spot, the focusing spot is limited in processing precision due to diffraction limit, and a single-beam laser direct writing lithography system is low in processing speed and cannot meet the requirements of actual production and application.
There is therefore a need for a high-throughput super-diffraction-limited focal spot to achieve ultra-fast high resolution for microscopic imaging and laser direct-write lithography.
Disclosure of Invention
The invention aims to provide a method for generating high-flux focal spots with super-diffraction limit, which can generate a focal spot array with super-diffraction limit, thereby carrying out stimulated emission loss microscopic imaging or laser direct writing photoetching in parallel and greatly improving the imaging and photoetching speed.
In order to achieve the above object, the present invention provides a method of generating a high-flux super-diffraction-limited focal spot, comprising the steps of:
1) generating exciting light;
2) modulating the generated loss light into hollow loss light;
3) combining the excitation light and the hollow loss beam and then converting the combined beam into a light beam array;
4) and focusing the light beam array on the sample to perform stimulated emission loss to generate a high-flux super-diffraction limit focal spot scanning sample.
In order to make the light intensity distribution of the light spot projected on the sample more uniform, it is preferable that the excitation light and the hollow loss light are converted into circularly polarized light and then the sample is scanned.
Preferably, the array of light beams is a square array. Such as a square or rectangular array.
Preferably, the array of collected light beams excites fluorescence emitted by the sample for microscopic imaging.
Preferably, the lithography is performed with a high-flux super-diffraction-limited focal spot focused by an array of beams on the sample.
The device can be used for realizing the method, the exhausted light emitted by the exhausted laser is modulated into a hollow beam by using a vortex phase plate, the hollow beam is combined with the excited light emitted by the excited laser, then the hollow beam is divided into a beam array by a plurality of beam splitters, and finally the beam array is focused on a sample surface to generate the high-flux super-diffraction-limit focal spot array, so that the speed of the stimulated emission loss microscopy and the laser direct writing lithography is greatly improved.
An apparatus for generating a high-flux super-diffraction-limited focal spot, comprising:
an excitation light source for emitting excitation light;
a loss light source for emitting loss light;
a modulator for modulating the loss light into hollow loss light;
the beam splitting system is used for combining the excitation light and the hollow loss light and then converting the combined light into a light beam array;
and the microscope system is used for focusing the light beam array on the sample to perform stimulated emission loss so as to generate a high-flux super-diffraction limit focal spot scanning sample.
Preferably, the excitation light source and the loss light source are divided into a plurality of groups, and each group comprises an excitation light source and a loss light source.
Preferably, the beam splitting system is correspondingly arranged on the light path of each group of the excitation light source and the loss light source, and comprises the following components in sequence:
a first beam splitter for combining the excitation light and the hollow loss light;
the second beam splitter is used for splitting the combined light beam into a plurality of light beams with equal intensity;
a 4f system consisting of a pair of lenses for demagnifying the plurality of equal intensity beams.
Preferably, the modulator is a vortex phase plate.
Preferably, the device further comprises a detector array, which is adapted to the light beam array and is used for collecting the emitted fluorescence for microscopic imaging.
The device mainly comprises an excitation system for generating exciting light, a loss system for generating loss light, a beam splitting system for generating a light beam array, a microscopic system for imaging and photoetching a sample and a detection system for emitting a fluorescence signal by the sample.
On the optical axis of arousing the system, be equipped with in proper order:
an excitation laser (excitation light source) for generating excitation light;
a collimating objective lens for collimating the excitation light emitted by the excitation laser;
and the half wave plate and the quarter wave plate are used for converting the collimated exciting light into circularly polarized exciting light.
On the optical axis of the loss light path, be equipped with in proper order:
a lossy laser (lossy light source) for generating lossy light;
a collimating objective lens for collimating the evanescent light emitted by the evanescent laser;
a vortex phase plate for converting the collimated lost light into hollow lost light;
and the half wave plate and the quarter wave plate are used for converting the hollow core loss light into circularly polarized hollow core loss light.
On the optical axis of the beam splitting optical path, are sequentially provided
A beam splitter for combining the circularly polarized excitation light and the circularly polarized hollow loss beam;
a beam splitter for splitting the combined beam into a plurality of equal intensity beams;
a 4f system consisting of a pair of lenses for demagnifying the plurality of equal intensity beams;
and the reflecting mirror is used for reflecting the shrunk light beams to the dichroic mirror to form an array.
On the optical axis of the microscope system, are sequentially arranged:
a dichroic mirror for reflecting excitation light and loss light and transmitting fluorescence;
the two-dimensional scanning galvanometer system is used for changing the azimuth angle of incident light and deflecting the light path so as to perform two-dimensional scanning and de-scanning on the sample;
the scanning lens is used for eliminating the distortion of the excitation light and the loss light after passing through the scanning galvanometer system, collimating and converging the fluorescence passing through the field lens and enabling the galvanometer and the entrance pupil surface of the objective lens to be conjugated;
the field lens is used for collimating and expanding the exciting light and the loss light passing through the scanning lens, conjugating the galvanometer and the entrance pupil surface of the objective lens and focusing the fluorescence passing through the objective lens;
the objective lens is used for focusing the excitation light and the loss light collimated by the field lens to a sample and collecting a fluorescent signal emitted by the sample on the sample stage;
the sample stage is used for placing a sample to be tested.
On the optical axis of detecting system, be equipped with in proper order:
the optical filter is used for filtering stray light transmitted by the dichroic mirror;
the focusing lens is used for focusing the fluorescent light beams passing through the optical filter onto an optical fiber array consisting of multimode optical fibers;
a detector array for acquiring the fluorescence signal.
Preferably, the wavelength of the excitation light is 440nm, and the wavelength of the loss light is 532 nm.
Preferably, the beam array, the multimode fiber array and the detector array are 10 by 10 square arrays.
Preferably, the two-dimensional scanning galvanometer system is a three-mirror galvanometer system to suppress scanning distortion, effectively fold the length of an optical path and ensure the compactness of the system structure.
Preferably, the detector is an Avalanche Photodiode (APD);
preferably, the objective lens has a Numerical Aperture (NA) of 1.4;
the principle of the invention is as follows:
the excitation laser emits excitation light, and the excitation light is converted into circularly polarized excitation light under the action of the half-wave plate and the quarter-wave plate after being collimated; the loss laser emits loss light, the loss light is converted into hollow loss light through a vortex phase plate after being collimated, then the hollow loss light is converted into circular polarization hollow loss light under the action of a half wave plate and a quarter wave plate, the circular polarization excitation light and the circular polarization hollow loss light are combined by using a beam splitter, then the light is split by the beam splitter and reflected to a dichroic mirror to generate a light beam array, then high-flux super-diffraction limit focal spots are formed under the modulation of a two-dimensional scanning galvanometer system and projected on a sample to be detected for two-dimensional scanning, and stimulated emission loss microscopic imaging or laser direct writing photoetching is carried out; finally, the high-flux super-diffraction limit fluorescence emitted by the sample is received by the detector array through the multimode fiber array.
Compared with the prior art, the invention has the following advantages:
(1) the method has extremely high stimulated emission loss microscopic imaging speed and laser direct writing photoetching speed;
(2) ultra-high resolution down to tens of nanometers;
drawings
FIG. 1 is a schematic diagram of the present invention for producing a high-throughput super-diffraction-limited focal spot;
FIG. 2 is a schematic diagram of an apparatus for generating a high throughput super-diffraction limited focal spot according to the present invention;
FIG. 3 is a schematic diagram of the arrangement of the multimode fiber array and the detector array according to the present invention;
fig. 4 is a schematic diagram of a high-flux super-diffraction-limit focal spot array in the present invention, wherein a is a schematic diagram of a solid excitation focal spot array, B is a schematic diagram of a hollow loss focal spot array, and C is a schematic diagram of a generated high-flux super-diffraction-limit focal spot array.
Detailed Description
A device for generating high-flux super-diffraction limit focal spots is schematically shown in fig. 1 and comprises ten layers of lasers, wherein each layer of lasers comprises an excitation laser and a loss laser, each layer of lasers generates a high-flux super-diffraction limit focal spot through beam splitting, and finally a high-flux super-diffraction limit focal spot array consisting of the ten high-flux super-diffraction limit focal spots is obtained.
The generating device of each high-flux super-diffraction limit focal spot is shown in fig. 2 and comprises: the optical fiber scanning device comprises an excitation laser 1, a first single-mode optical fiber 2, a first collimating lens 3, a first quarter wave plate 4, a first quarter wave plate 5, a first beam splitter 6, a loss laser 7, a second single-mode optical fiber 8, a second collimating lens 9, a vortex phase plate 10, a second half wave plate 11, a second quarter wave plate 12, a first reflecting mirror 13, a second beam splitter to tenth beam splitters 14-22, a second reflecting mirror 23, first to tenth focusing lenses 24-33, first to tenth diverging lenses 34-43, third to eleventh reflecting mirrors 44-52, a tenth reflecting mirror 53, a dichroic mirror 54, a two-dimensional scanning galvanometer system 55, a scanning lens 56, a field lens 57, a thirteenth reflecting mirror 58, an objective lens 59, a sample stage 60, a filter 61, an eleventh focusing lens 62, a multimode optical fiber array 63 and a detector array 64.
The device embodiment of the invention is mainly divided into four parts: an excitation system for generating excitation light, a depletion system for generating depletion light, a beam splitting system for generating a beam array, a microscopic system for imaging and lithography of a sample and a detection system for collecting a fluorescence signal emitted by the sample.
The excitation laser 1, the first single-mode fiber 2, the first collimating lens 3, the first one-half wave plate 4, the first one-quarter wave plate 5 and the first beam splitter 6 are sequentially arranged on an optical axis of the excitation system;
the loss laser 7, the second single-mode fiber 8, the second collimating lens 9, the vortex phase plate 10, the second half-wave plate 11, the second quarter-wave plate 12 and the first reflector 13 are sequentially arranged on an optical axis of the loss system;
the second beam splitter to the tenth beam splitter 14 to 22, the second reflecting mirror 23, the first to tenth focusing lenses 24 to 33, the first to tenth diverging lenses 34 to 43, the third to eleventh reflecting mirrors 44 to 52, and the tenth reflecting mirror 53 are sequentially disposed on the optical axis of the beam splitting system.
Wherein, the dichroic mirror 54, the two-dimensional scanning galvanometer system 55, the scanning lens 56, the field lens 57, the thirteenth reflecting mirror 58, the objective lens 59 and the sample stage 60 are sequentially arranged on the optical axis of the microscope system;
the filter 61, the eleventh focusing lens 62, the multimode fiber array 63 and the detector array 64 are sequentially arranged on the optical axis of the detection system;
with the apparatus shown in fig. 2, the method for generating a high-throughput super-diffraction-limited focal spot was used as follows:
1) excitation light emitted by the excitation laser 1 (in this embodiment, laser with a wavelength of 440nm is used as the excitation light) is coupled into the first single-mode fiber 2, then is emitted from the first single-mode fiber 2, is collimated by the first collimating lens 3, is modulated into circularly polarized excitation light by the first half-wave plate 4 and the first quarter-wave plate 5, and then reaches the first beam splitter 6;
2) the loss laser 7 emits loss light (in this embodiment, laser with a wavelength of 532nm is used as the loss light), the loss light is coupled into the second single-mode fiber 8, then the loss light is emitted from the second single-mode fiber 8 and collimated by the second collimating lens 9, then the loss light becomes hollow loss light through the vortex phase plate 10, the hollow loss light is circularly polarized hollow loss light by using the second half-wave plate 11 and the second quarter-wave plate 12, and then the hollow loss light is reflected to the first beam splitter 6 by the first reflector 13;
3) the first beam splitter 6 combines the circularly polarized excitation light and the circularly polarized hollow loss light, the combined light beam is split into ten light beams with equal light intensity by the second beam splitter to the tenth beam splitter, the light beams respectively pass through ten 4f systems consisting of first to tenth focusing lenses 24-33 and first to tenth diverging lenses 34-43 and then are contracted, and finally the light beams are reflected to a dichroic mirror 54 by a second reflecting mirror 23 and third to tenth reflecting mirrors 44-53 to generate a light beam array;
4) the light beam array is reflected by the dichroic mirror 54 to reach the two-dimensional scanning galvanometer system 55, the two-dimensional scanning galvanometer system 55 changes the azimuth angle of the incident light beam array and deflects the light path, the light beam array emitted by the two-dimensional scanning galvanometer system 55 is subjected to distortion elimination after passing through the scanning lens 56, is subjected to collimation and beam expansion by the field lens 57, is reflected to the objective lens 59 by the thirteenth reflecting mirror 58, is focused on a sample placed on the sample table 60 through the objective lens 59 to be subjected to stimulated emission loss to generate a high-flux super-diffraction limit focal spot scanning sample, and is subjected to microscopic imaging or laser direct writing photoetching;
5) the high-flux super-diffraction limit fluorescence emitted by the sample is received by the detector array through the multimode fiber array
The high-flux super-diffraction limit fluorescent signal emitted by the sample is collected by the objective lens 59, then reflected to the field lens 57 by the thirteenth reflecting mirror 58, focused by the field lens 57 and collimated by the scanning lens 56 to reach the scanning galvanometer system 55, de-scanned, transmitted to the filter 61 by the dichroic mirror 54, filtered to remove laser and fluorescence with other wavelengths, and focused on the multimode fiber array 63 by the eleventh focusing lens 62. Finally, the detector array 64 is used for receiving high-flux super-diffraction limit fluorescence signals emitted by the sample in a two-dimensional scanning process in parallel.
FIG. 3 shows the arrangement of multimode fiber array and detector array, where one hundred multimode fibers form a square multimode fiber array and one hundred APD detectors form a square detector array.
Fig. 4 is a schematic diagram of a high-flux super-diffraction-limit focal spot array in the present invention, wherein a is a schematic diagram of a solid excitation focal spot array, B is a schematic diagram of a hollow loss focal spot array, and C is a schematic diagram of a generated high-flux super-diffraction-limit focal spot array. . It can be seen that the right image is smaller, that is to say higher resolution, than the full width half maximum of the left image.
The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of generating a high-flux, super-diffraction-limited focal spot, comprising the steps of:
1) generating exciting light;
2) modulating the generated loss light into hollow loss light;
3) combining the excitation light and the hollow loss beam and then converting the combined beam into a light beam array;
4) and focusing the light beam array on the sample to perform stimulated emission loss to generate a high-flux super-diffraction limit focal spot scanning sample.
2. The method for generating a high-throughput super-diffraction-limited focal spot according to claim 1, wherein the excitation light and the hollow loss light are converted into circularly polarized light and then the sample is scanned.
3. The method of generating a high flux super-diffraction limited focal spot according to claim 1, wherein the array of light beams is a square array.
4. The method for generating high-throughput super-diffraction-limited focal spots according to claim 1, wherein fluorescence emitted from the sample is excited by the collection beam array and is imaged microscopically.
5. The method of generating high-throughput super-diffraction-limited focal spots according to claim 1, wherein the high-throughput super-diffraction-limited focal spot focused on the sample by the array of light beams is used for lithography.
6. An apparatus for generating a high flux super-diffraction limited focal spot, comprising:
an excitation light source for emitting excitation light;
a loss light source for emitting loss light;
a modulator for modulating the loss light into hollow loss light;
the beam splitting system is used for combining the excitation light and the hollow loss light and then converting the combined light into a light beam array;
and the microscope system is used for focusing the light beam array on the sample to perform stimulated emission loss so as to generate a high-flux super-diffraction limit focal spot scanning sample.
7. The device for generating high-throughput super-diffraction-limited focal spots according to claim 6, wherein the excitation light source and the depletion light source are divided into a plurality of groups, each group comprising an excitation light source and a depletion light source.
8. The apparatus according to claim 6 or 7, wherein the beam splitting system is disposed in the optical path of each of the excitation light source and the loss light source, and comprises:
a first beam splitter for combining the excitation light and the hollow loss light;
the second beam splitter is used for splitting the combined light beam into a plurality of light beams with equal intensity;
a 4f system consisting of a pair of lenses for demagnifying the plurality of equal intensity beams.
9. The device for generating a high flux super diffraction limited focal spot according to claim 6, wherein said modulator is a vortex phase plate.
10. The apparatus for generating high flux super-diffraction limited focal spots according to claim 6 further comprising a detector array adapted to the array of light beams for collecting the emitted fluorescence for microscopic imaging.
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Application publication date: 20201103 |