CN110632045B - Method and device for generating parallel super-resolution focal spots - Google Patents

Method and device for generating parallel super-resolution focal spots Download PDF

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CN110632045B
CN110632045B CN201910855174.9A CN201910855174A CN110632045B CN 110632045 B CN110632045 B CN 110632045B CN 201910855174 A CN201910855174 A CN 201910855174A CN 110632045 B CN110632045 B CN 110632045B
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light
loss
excitation
polarization
loss light
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CN110632045A (en
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匡翠方
陈宇宸
刘旭
郝翔
李海峰
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Zhejiang University ZJU
Zhejiang Lab
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Zhejiang University ZJU
Zhejiang Lab
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21Polarisation-affecting properties
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6402Atomic fluorescence; Laser induced fluorescence
    • GPHYSICS
    • G02OPTICS
    • G02BOPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58Optics for apodization or superresolution; Optical synthetic aperture systems
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21Polarisation-affecting properties
    • G01N2021/216Polarisation-affecting properties using circular polarised light

Abstract

The invention discloses a method for generating parallel super-resolution focal spots, which specifically comprises the following steps: modulating excitation light emitted by a laser into a multi-focus Gaussian light spot by using a spatial light modulator; depletion light emitted by the laser is divided into four beams of depletion light by the polarization beam splitter and the two gratings in the vertical direction, and the four beams of depletion light are interfered at the focal plane behind the objective lens to generate a multi-focus hollow light spot; the multifocal hollow depletion light inhibits fluorescent molecules excited by the outer ring of the multifocal Gaussian excitation light from emitting fluorescence, so that effective fluorescent signals far smaller than a diffraction limit are obtained in parallel to perform microscopic imaging and laser direct writing photoetching. The invention also discloses a device for generating the parallel super-resolution focal spots. The invention can realize the stimulated emission loss microscopic imaging and the laser direct-writing photoetching processing with ultrahigh speed and ultrahigh resolution.

Description

Method and device for generating parallel super-resolution focal spots
Technical Field
The invention belongs to the field of optical engineering, and particularly relates to a method and a device for generating parallel super-resolution focal spots.
Background
With the continuous development of the scientific and technical level, the requirements of people on observation and control means in the micro world are continuously increased, so that the development of optical sensing and control capability on the sub 50 nanometer scale is urgently needed. The stimulated emission depletion technology is the most common technical means for optical sensing and manipulation to break through the diffraction limit. However, the stimulated emission depletion technology relies on a single-point scanning method, and the operation speed of the technology is greatly limited. Therefore, there is a need for a technique that can generate multiple super-resolution focal spots to achieve fast ultra-high resolution microscopic imaging and laser direct-write lithography in parallel.
Disclosure of Invention
The object of the present invention is to provide a method for generating parallel super-resolution focal spots, with which the speed of the stimulated emission depletion technique can be significantly increased.
The device can be used for realizing the method, the excitation light emitted by the excitation laser is divided into n solid excitation light spots by using the spatial light modulator, the loss light emitted by the loss laser is divided into four light beams, the four light beams are interfered on the back focal plane to generate corresponding n hollow loss light spots, the stimulated emission loss fluorescence excited by the sample is respectively received by the corresponding n detectors after microscopic imaging and laser direct writing photoetching, and the imaging speed of the common stimulated emission loss technology can be greatly improved.
In order to achieve the above object, the present invention provides a method for generating parallel super-resolution focal spots, comprising the steps of:
1) the excitation laser emits excitation light, the excitation light is converted into linear polarization excitation light after being collimated, and a spatial light modulator is used for carrying out phase modulation on the linear polarization excitation light to generate n Gaussian excitation light beams;
2) converting the phase-modulated linear polarization exciting light into circular polarization exciting light by using a quarter-wave plate, wherein the circular polarization exciting light is projected on a sample to be measured under the modulation of a two-dimensional scanning galvanometer system to carry out two-dimensional scanning;
3) the loss laser emits loss light, the loss light is collimated and then divided into two beams of linear polarization loss light through the polarization beam splitter, and the two beams of linear polarization loss light respectively pass through the grating to finally generate four beams of linear polarization loss light;
4) the four-beam polarization loss light is converted into circularly polarized light after passing through a quarter-wave plate, and is focused on a sample to be detected to generate interference through a two-dimensional scanning galvanometer system to form n hollow loss light spots;
5) the loss light spots loss the fluorescent molecules excited by the outer ring of the Gaussian excitation light spots in advance, so that n effective stimulated emission loss fluorescent signals with smaller full width at half maximum are obtained for microscopic imaging and laser direct writing photoetching, and n detectors are used for receiving n effective fluorescent signals emitted by the sample to be detected in the two-dimensional scanning process.
The linear polarization exciting light is p-polarized light, and the spatial light modulator can only modulate the p-polarized light.
The linear polarization exciting light is converted into circular polarization light, and then the sample is scanned so that the light intensity distribution of light spots projected onto the sample is more uniform.
Wherein, the scanning range of the two-dimensional scanning galvanometer system is set according to the required field of view.
And the polarization states of the two beams of linear polarization loss light passing through the polarization beam splitter are perpendicular to each other.
Wherein the excitation light spots and the loss light spots are aligned one by one.
The principle of the invention is as follows:
the excitation laser emits excitation light, the excitation light is converted into linearly polarized excitation light after being collimated, and a spatial light modulator is used for carrying out phase modulation on the linearly polarized excitation light to generate a plurality of Gaussian excitation light beams; converting the phase-modulated linear polarization exciting light into circular polarization exciting light by using a quarter-wave plate, and then projecting the circular polarization exciting light on a sample to be measured under the modulation of a two-dimensional scanning galvanometer system to perform two-dimensional scanning; the loss laser emits loss light, the loss light is converted into circularly polarized light under the action of a half wave plate and a quarter wave plate after being collimated, the circularly polarized light is divided into two beams of linear polarization loss light through a polarization beam splitter, then the circularly polarized loss light is converted into four beams of linear polarization loss light through a grating, the circularly polarized loss light passes through a two-dimensional scanning galvanometer system, the interference is generated on a sample to be detected by focusing, a plurality of hollow loss light spots are formed, the outer ring loss of Gaussian excitation light spots is lost, and a plurality of stimulated emission loss fluorescent signals with smaller full width at half maximum are obtained to carry out microscopic imaging and laser direct writing photoetching. And finally, receiving stimulated emission depletion fluorescence signals emitted by the sample to be detected by using a plurality of detectors.
To achieve the above-mentioned another object, the present invention further provides an apparatus for generating parallel super-resolution focal spots, comprising an excitation system for generating excitation light, a depletion system for generating depletion light, a microscope system for imaging and lithography of a sample, and a detection system for collecting fluorescence signals emitted from the sample.
On the optical axis of excitation light path, be equipped with in proper order:
an excitation laser for generating excitation light;
a collimating objective lens for collimating the excitation light emitted by the excitation laser;
a polarizer for converting the collimated excitation light into linearly polarized excitation light;
a half wave plate for converting the linearly polarized excitation light into p-polarized excitation light;
the spatial light modulator is used for carrying out phase modulation on the p-polarized exciting light to generate n Gaussian exciting light spots; preferably 100 gaussian excitation spots are generated;
the quarter wave plate is used for converting the n p-polarized Gaussian excitation light spots into circularly polarized excitation light;
a dichroic mirror for transmitting the excitation light;
on the optical axis of the loss light path, be equipped with in proper order:
a lossy laser for generating lossy light;
a collimating objective lens for collimating the evanescent light emitted by the evanescent laser;
polarizer for converting the collimated lost light into linearly polarized lost light
A half wave plate for adjusting the intensity of the linear polarization loss light;
a quarter wave plate for converting the loss light with the adjusted light intensity into loss light with circular polarization;
the polarization beam splitter is used for splitting the circular polarization loss light into a beam of p-polarization loss light and a beam of s-polarization loss light;
a grating for splitting the p-polarization loss light into two beams of p-polarization loss light;
a grating for splitting the s-polarization loss light into two s-polarization loss lights;
a polarization beam splitter for combining the two beams of p-polarization loss light and the two beams of s-polarization loss light;
a dichroic mirror for reflecting the four-beam polarization-lost light;
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, enabling the four-beam polarization loss light to interfere to generate 100 loss light spots and collecting a fluorescence 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 100 multimode optical fibers;
and the detector array consists of 100 detectors for acquiring the fluorescence signals.
The controller is used for controlling the laser, the spatial light modulator and the scanning galvanometer system, and the computer is used for processing the fluorescence signal;
the angle between the incident light and the emergent light of the spatial light modulator should be less than 5 degrees to reduce the crosstalk effect caused by light passing through more than one pixel region and to make the phase travel approach the design value.
Preferably, the wavelength of the excitation light is 440nm, and the wavelength of the loss light is 532 nm.
Preferably, the excitation and loss spots, multimode fiber and detector array are a 10 by 10 square array.
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;
compared with the prior art, the invention has the following advantages:
(1) ultra-high resolution down to sub-50 nm;
(2) the method has extremely high microscopic imaging and laser direct writing photoetching speed far exceeding the common stimulated emission loss technology;
drawings
FIG. 1 is a schematic view of an apparatus of the present invention;
FIG. 2 is a schematic view of an excitation spot array according to the present invention;
FIG. 3 is a schematic diagram of a four-beam lossy light incident objective lens of the present invention;
FIG. 4 is a schematic diagram of a loss spot array in accordance with the present invention;
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but the present invention is not limited thereto.
An apparatus for generating parallel super-resolution focal spots, as shown in fig. 1, comprises: the device comprises an excitation laser 1, a first single-mode optical fiber 2, a first collimating lens 3, a first polarizer 4, a first one-half wave plate 5, a spatial light modulator 6, a first reflector 7, a first one-quarter wave plate 8, a first dichroic mirror 9, a second dichroic mirror 10, a two-dimensional scanning galvanometer system 11, a scanning lens 12, a field lens 13, a second reflector 14, an objective lens 15, a sample stage 16, a loss laser 17, a second single-mode optical fiber 18, a second collimating lens 19, a second polarizer 20, a second one-half wave plate 21, a second one-quarter wave plate 22, a first polarization beam splitter 23, a first grating 24, a third reflector 25, a second grating 26, a fourth reflector 27, a second polarization beam splitter 28, a filter 29, a focusing lens 30, a multimode fiber array 31, a detector array 32 and a computer 33.
The device embodiment of the invention is mainly divided into four parts: an excitation system for generating excitation light, an evanescent system for generating evanescent light, a microscopic system for imaging and lithography of the sample, a detection system for collecting fluorescence signals emitted by the sample, and a processor, in this embodiment a computer 34.
The excitation laser 1, the first single-mode fiber 2, the first collimating lens 3, the first polarizer 4, the first quarter-wave plate 5, the spatial light modulator 6, the first reflector 7, the first quarter-wave plate 8 and the first dichroic mirror 9 are sequentially arranged on an optical axis of the excitation system;
the loss laser 17, the second single-mode fiber 18, the second collimating lens 19, the second polarizer 20, the second half-wave plate 21, the second quarter-wave plate 22, the first polarization beam splitter 23, the first grating 24, the third reflector 25, the second grating 26, the fourth reflector 27, the second polarization beam splitter 28 and the first dichroic mirror 9 are sequentially arranged on the optical axis of the loss system;
the second dichroic mirror 10, the two-dimensional scanning galvanometer system 11, the scanning lens 12, the field lens 13, the second reflecting mirror 14, the objective lens 15 and the sample stage 16 are sequentially arranged on an optical axis of the microscope system;
wherein, the filter 29, the focusing lens 30, the multimode fiber array 31 and the detector array 32 are sequentially arranged on the optical axis of the detection system;
wherein, the computer 33 is used for controlling the laser states output by the excitation laser 1 and the loss laser 17, the modulation pattern switching of the spatial light modulator 6, the scanning of the two-dimensional scanning galvanometer system 11 and the signal acquisition of the detector array 32;
with the apparatus shown in fig. 1, the method of generating parallel super-resolution focal spots is used as follows:
1) excitation light emitted by the excitation laser 1 (in this embodiment, laser with a wavelength of 440nm is used as excitation light) is coupled into the first single-mode fiber 2, then is emitted from the first single-mode fiber 2 and collimated by the first collimating lens 3, and then becomes linear polarization excitation light by the first polarizer 4, the linear polarization excitation light is modulated into p-polarization excitation light by the half-wave plate 5, and then reaches the spatial light modulator 6 for phase modulation, so as to generate n gaussian excitation light beams as shown in fig. 2, wherein the central gaussian excitation spot is shown in a white box of fig. 2;
2) the first reflector 7 reflects the modulated p-polarized excitation light to the first quarter-wave plate 8, the first quarter-wave plate 8 converts the phase-modulated p-polarized excitation light into circularly polarized excitation light, the circularly polarized excitation light passes through the transmission of the first dichroic mirror 9 and the reflection of the second dichroic mirror 10 and reaches the two-dimensional scanning galvanometer system 11, the two-dimensional scanning galvanometer system 11 changes the azimuth angle of the incident circularly polarized excitation light and deflects the light path, the circularly polarized excitation light emitted by the two-dimensional scanning galvanometer system passes through the scanning lens 12 to eliminate distortion, and then passes through the collimation and beam expansion of the field lens 13, and is reflected to the objective lens 15 by the second reflector 14, and finally is focused to a sample to be measured on the sample stage 16 through the objective lens 15;
3) the loss laser 17 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 18, and then the loss light is emitted from the second single-mode fiber 18, collimated by the second collimating lens 19, and then becomes linear polarization loss light by the second polarizer 20, and the linear polarization loss light passes through after the intensity of the linear polarization loss light is adjusted by the second half-wave plate 21, the second quarter-wave plate 22 is modulated into circular polarization loss light, and then divided into two linear polarization loss lights with mutually perpendicular polarization states by the first polarization beam splitter 23, one linear polarization loss light is divided into two beams by the first grating 24 arranged transversely and then reflected to the second polarization beam splitter 28 by the third reflector 25, and the other beam of polarization loss light is divided into two beams by the second grating 26 arranged longitudinally and then reflected to the second polarization beam splitter 28 by the fourth reflector 27;
4) the two pairs of linear polarization loss light (one pair of linear polarization hollow loss light polarization is along the x-axis direction, the other pair is along the y-axis direction) combined by the second polarization beam splitter 28 reach the two-dimensional scanning galvanometer system 11 through the reflection of the first dichroic mirror 9 and the second dichroic mirror 10, the two-dimensional scanning galvanometer system 11 changes the azimuth angles of the two pairs of incident linear polarization loss light and deflects the light path, the two pairs of linear polarization loss light emitted by the two-dimensional scanning galvanometer system eliminates distortion after passing through the scanning lens 12, then is collimated and expanded by the field lens 13, is reflected onto the objective lens 15 by the second reflecting mirror 14, and finally is focused onto a sample to be measured on the sample stage 16 through the objective lens 15 to generate interference (two pairs of linear polarization hollow loss light) as shown in fig. 3, so as to form n hollow loss light beams as shown in fig. 4, wherein the most central hollow loss light spot is represented in a white square frame in fig. 4;
5) the n hollow loss light spots loss the fluorescent molecules excited by the outer rings of the n Gaussian excitation light spots in advance, so that n effective stimulated emission loss fluorescent signals with higher resolution are obtained, the effective stimulated emission loss fluorescent signals are collected by the objective lens 15 after microscopic imaging or laser direct writing photoetching is carried out on a sample, then are reflected to the field lens by the second reflecting mirror 14, reach the scanning galvanometer system 11 through focusing of the field lens 13 and collimation of the scanning lens 12, are transmitted to the filter 29 by the second dichroic mirror 10 after being subjected to de-scanning, and are focused on the multimode optical fiber array 31 by the focusing lens 30 after laser and fluorescence with other wavelengths are filtered. Finally, a detector array 32 composed of n detectors is used for receiving n effective fluorescence signals emitted by the sample to be detected in the two-dimensional scanning process in parallel.
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 (5)

1. A method of generating parallel super-resolution focal spots, comprising the steps of:
1) the exciting light is projected to a sample to be detected after phase modulation, and n solid exciting light spots are generated and fluorescence signals are excited;
2) the loss light is projected to a sample to be measured to form n hollow loss light spots in an interference mode, and the outer ring fluorescence of the solid excitation light spot corresponding to each hollow loss light spot is lost;
3) collecting the fluorescence signals after the outer ring fluorescence excited by the solid excitation light spots is lost by using n detectors to perform microscopic imaging and laser direct writing photoetching;
in the step 2), the loss light is split into four beam lines of polarized loss light, and the four beam lines of polarized loss light are focused on a sample to be detected to generate interference, so that the n hollow loss light spots are formed;
the specific steps of splitting the loss light into four-beam polarization loss light are as follows:
the loss light is converted into linear polarization loss light after being collimated, is modulated into circular polarization loss light through a quarter-wave plate, and is split into two beams of linear polarization loss light with mutually vertical polarization states;
the loss light is converted into two beams of linear polarization loss light with mutually vertical polarization states after primary beam splitting, and the two beams of linear polarization loss light are split again respectively to form four beams of linear polarization loss light.
2. The method for generating parallel super-resolution focal spots according to claim 1, wherein in step 1), the excitation light is converted into linearly polarized excitation light after being collimated, the linearly polarized excitation light is phase-modulated by using a spatial light modulator to generate n gaussian excitation beams, and after being projected onto a sample to be measured, n solid excitation spots are generated.
3. A device for generating parallel super-resolution focal spots comprises an excitation system for generating excitation light, a loss system for generating loss light, a microscopic system for imaging and photoetching of a sample, a detection system for collecting a fluorescence signal emitted by the sample and a processor; the method is characterized in that:
the excitation system comprises an excitation laser for emitting excitation light and a spatial light modulator for carrying out phase modulation on the excitation light, the excitation light after phase modulation is projected to a sample to be measured through a microscope system to generate n solid excitation light spots and excite a fluorescence signal;
the loss system comprises a loss laser for emitting loss light and a beam splitting assembly for splitting the loss light into four beams of polarized loss light, the four beams of polarized loss light are projected onto a sample to be detected through a microscope system to generate interference to form n hollow loss light spots, the outer ring fluorescence loss of the solid excitation light spot corresponding to each hollow loss light spot is lost, and microscopic imaging and laser direct writing photoetching are carried out;
the detection system comprises n detectors for respectively collecting the fluorescence signals after the loss of the outer ring fluorescence excited by each solid excitation light spot;
the loss system comprises, arranged in the path of the loss light:
a second collimating lens;
a second polarizer for converting the lost light into linearly polarized lost light;
a second half wave plate for adjusting the intensity of the linearly polarized lost light;
the second quarter-wave plate is used for converting the linear polarization loss light into circular polarization loss light;
the beam splitting assembly comprises:
the first polarization beam splitter is used for splitting the circular polarization loss light into two beams of linear polarization loss light with mutually vertical polarization states;
the first grating is used for dividing one beam of linear polarization loss light into two beams of polarization loss light;
the second grating is vertical to the first grating in direction and is used for dividing the other bunch of polarization loss light into two bunches of polarization loss light;
and the second polarization beam splitter is used for combining the four beam lines of polarization loss light into the same optical path.
4. The apparatus for generating parallel super-resolution focal spots according to claim 3, wherein the excitation system further comprises, disposed in the excitation light path:
a first collimating lens;
a first polarizer for converting the excitation light into linearly polarized excitation light;
the first one-half wave plate is used for modulating the linear polarization excitation light into p-polarization excitation light and then entering the spatial light modulator;
the first quarter-wave plate is used for converting the p-polarized exciting light after phase modulation into circular polarized exciting light.
5. The apparatus for generating parallel super-resolution focal spots according to claim 3, wherein the microscope system comprises a two-dimensional scanning galvanometer system, a scanning lens, a field lens, an objective lens and a sample stage arranged in sequence along an optical axis;
the detection system comprises a filter, a focusing lens, a multimode fiber array and a detector array which are sequentially arranged along an optical axis; the detector array includes a number of detectors equal to the number of n solid excitation spots.
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