CN111879737A - Device and method for generating high-flux super-diffraction limit focal spot - Google Patents

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

A device and method for generating a high-throughput ultra-diffraction-limited focal spot

technical field

The invention belongs to the field of optical engineering, and particularly relates to a device and method for generating a high-flux ultra-diffraction-limited focal spot.

Background technique

Due to the diffraction limit, the resolution of far-field optical microscopy has been essentially limited to half the wavelength of the excitation light since the early 20th century. While electron and scanning probe microscopes provide finer detail, they cannot non-invasively image living cells and tissues and other transparent materials in three dimensions. Since then, various far-field super-resolution microscopy imaging methods have been proposed, among which stimulated emission depletion microscopy is the most developed and widely used.

Stimulated emission depletion microscopy mainly uses a hollow depletion beam to cover the diffraction-limited excitation beam, so that the excited fluorophores in the outer ring of the spot return to the ground state instantaneously, while the excited fluorescent molecules in the center of the spot are normal. The emitted fluorescence is received as the effective signal, resulting in a resolution well beyond the diffraction limit. However, stimulated emission depletion microscopy is imaging in the form of single-point scanning, which requires a long imaging time, which limits the microscopic imaging in a large field of view. photobleaching.

At the same time, the laser direct writing lithography technology acts on the sample material by focusing the spot, and the focusing spot will also limit the processing accuracy due to the diffraction limit. Moreover, the processing speed of the single-beam laser direct writing lithography system is slow, which cannot reach the actual production and application requirements.

Therefore, a high-throughput ultra-diffraction-limited focal spot is needed to achieve ultra-fast and high-resolution microscopic imaging and laser direct writing lithography.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for generating a high-throughput ultra-diffraction-limited focal spot, by which an ultra-diffraction-limited focal spot array can be generated, so as to perform stimulated emission loss microscopic imaging or laser direct writing lithography in parallel, Greatly increases the speed of imaging and lithography.

In order to achieve the above object, the method for generating a high-throughput ultra-diffraction-limited focal spot provided by the present invention comprises the following steps:

1) generate excitation light;

2) modulate the generated loss light into hollow loss light;

3) Combine the excitation light and the hollow loss light into a beam array;

4) Focus the beam array on the sample for stimulated emission loss to generate a high-throughput ultra-diffraction-limited focal spot to scan the sample.

In order to make the light intensity distribution of the light spot projected on the sample more uniform, preferably, both the excitation light and the hollow loss light are converted into circularly polarized light and then the sample is scanned.

Preferably, the beam array is a square array. Such as square or rectangular arrays.

Preferably, the collection beam array excites the fluorescence emitted by the sample to perform microscopic imaging.

Preferably, the photolithography is performed using a high-throughput ultra-diffraction-limited focal spot where the beam array is focused on the sample.

Another object of the present invention is to provide a device for generating a high-throughput ultra-diffraction-limited focal spot that realizes the above method, and the device can be used to realize the above method, using a vortex phase plate to modulate the depletion light emitted by the depletion laser into The hollow beam is combined with the excitation light emitted by the excitation laser, and then divided into beam arrays by multiple beam splitters, and finally focused on the sample surface to generate a high-flux ultra-diffraction-limited focal spot array, which greatly improves the loss of stimulated emission. The speed of microtechnology and laser direct-write lithography.

A device for generating a high-throughput ultra-diffraction-limited focal spot, comprising:

Excitation light source for emitting excitation light;

A lossy light source for emitting lossy light;

a modulator for modulating lossy light into hollow lossy light;

Beam splitting system, used to combine excitation light and hollow loss light into beam array;

Microscopy system for SED-generated high-throughput ultra-diffraction-limited focal spot scanning of a sample by focusing an array of beams on a sample.

Preferably, the excitation light source and the loss light source are divided into multiple groups, and each group includes an excitation light source and a loss light source.

Preferably, the beam splitting system is correspondingly arranged on the optical path of each group of excitation light sources and loss light sources, including sequentially arranged:

a first beam splitter for combining excitation light and hollow loss light;

a second beam splitter for dividing the combined beam into a plurality of beams of equal intensity;

A 4f system consisting of a pair of lenses for condensing the plurality of beams of equal intensity.

Preferably, the modulator is a vortex phase plate.

Preferably, a detector array is also included, adapted to the beam array, for collecting the emitted fluorescence for microscopic imaging.

The device of the present invention mainly includes an excitation system for generating excitation light, a loss system for generating loss light, a beam splitting system for generating beam arrays, a microscope system for sample imaging and lithography, and a detection system for fluorescent signals emitted by the sample.

On the optical axis of the excitation system, there are:

Excitation lasers (excitation light sources) for generating excitation light;

a collimating objective lens for collimating the excitation light emitted by the excitation laser;

A half-wave plate and a quarter-wave plate for converting the collimated excitation light into circularly polarized excitation light.

On the optical axis of the loss optical path, there are:

lossy lasers (lossy light sources) for generating lossy light;

a collimating objective for collimating the lossy light emitted by the lossy laser;

a vortex phase plate for converting the collimated loss light into hollow loss light;

A half-wave plate and a quarter-wave plate for converting the hollow loss light into circularly polarized hollow loss light.

On the optical axis of the beam splitting optical path, there are sequentially

a beam splitter for combining the circularly polarized excitation light and the circularly polarized hollow loss light;

a beam splitter for dividing the combined beam into a plurality of beams of equal intensity;

a 4f system consisting of a pair of lenses for condensing the plurality of equal-intensity beams;

A mirror for reflecting the condensed light beam to a dichroic mirror to form an array.

On the optical axis of the microscope system, there are:

Dichroic mirrors for reflecting excitation and loss light and transmitting fluorescence;

A 2D scanning galvanometer system for changing the azimuth angle of the incident light and deflecting the light path to scan and de-scan the sample in 2D;

A scanning lens for eliminating the distortion of the excitation light and the loss light after passing through the scanning galvanometer system, collimating and condensing the fluorescence passing through the field lens, and making the galvanometer and the entrance pupil plane of the objective lens conjugate;

A field lens for collimating and expanding the excitation light and the loss light passing through the scanning lens, conjugating the entrance pupil plane of the galvanometer and the objective lens, and focusing the fluorescence passing through the objective lens;

an objective lens for focusing the excitation light and the depleted light collimated by the field lens to the sample, and collecting the fluorescent signal emitted by the sample on the sample stage;

Sample stage for placing the sample to be tested.

On the optical axis of the detection system, there are:

A filter for filtering out stray light transmitted by a dichroic mirror;

a focusing lens for focusing the fluorescent light beam passed through the filter onto an optical fiber array composed of multimode optical fibers;

A detector array for collecting the fluorescent signal.

Preferably, the wavelength of the excitation light is 440 nm, 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, so as to suppress scanning distortion, effectively fold the optical path length, and ensure the compactness of the system structure.

Preferably, the detector is an avalanche photodiode (APD);

Preferably, the numerical aperture (NA) of the objective lens is 1.4;

The principle of the present invention is as follows:

The excitation laser emits the excitation light, which is collimated and converted into circularly polarized excitation light under the action of the half-wave plate and the quarter-wave plate; the loss laser emits the loss light, which is collimated and passed through the vortex phase The plate becomes hollow loss light, which is then converted into circularly polarized hollow loss light under the action of a half-wave plate and a quarter-wave plate. The circularly polarized excitation light and the circularly polarized hollow loss light are combined by a beam splitter. The beam is then split by multiple beam splitters and then reflected to a dichromatic mirror to generate a beam array. Then, under the modulation of a two-dimensional scanning galvanometer system, a high-throughput ultra-diffraction-limited focal spot is formed and projected on the sample to be tested for two-dimensional scanning. Stimulated emission depletion microscopy imaging or laser direct writing lithography; finally, the high-throughput ultra-diffraction-limited fluorescence emitted by the sample is received by the detector array through the multimode fiber array.

Compared with the prior art, the present invention has the following advantages:

(1) It has extremely high stimulated emission loss microscopic imaging speed and laser direct writing lithography speed;

(2) Ultra-high resolution down to tens of nanometers;

Description of drawings

Fig. 1 is the schematic diagram that the present invention produces high flux ultra-diffraction limit focal spot;

2 is a schematic diagram of a device for generating a row of high-flux ultra-diffraction-limited focal spots in the present invention;

3 is a schematic diagram of the arrangement of the multimode fiber array and the detector array in the present invention;

Figure 4 is a schematic diagram of a high-throughput ultra-diffraction-limited focal spot array in the present invention, wherein Figure A is a schematic diagram of a solid excitation focal spot array, Figure B is a schematic diagram of a hollow loss focal spot array, and Figure C is a generated high-throughput ultra-diffraction-limited focal spot array Schematic diagram of the array.

Detailed ways

A device for generating high-flux ultra-diffraction-limited focal spots, the schematic diagram of which is shown in Figure 1, including ten layers of lasers, where each layer has an excitation laser and a loss laser, and each layer of lasers generates a row of high-flux lasers by beam splitting The ultra-diffraction-limited focal spot is finally obtained, and a high-throughput ultra-diffraction-limited focal spot array consisting of ten rows of high-throughput ultra-diffraction-limited focal spots is obtained.

The device for generating high-throughput ultra-diffraction-limited focal spots in each row is shown in Figure 2, including: an excitation laser 1, a first single-mode fiber 2, a first collimating lens 3, a first half-wave plate 4, a first A quarter-wave plate 5, a first beam splitter 6, a lossy laser 7, a second single-mode fiber 8, a second collimating lens 9, a vortex phase plate 10, a second half-wave plate 11, The second quarter wave plate 12, the first reflection mirror 13, the second beam splitter to the tenth beam splitter 14-22, the second reflection mirror 23, the first to tenth focusing lenses 24-33, the first To the tenth divergent lens 34-43, the third to eleventh mirrors 44-52, the twelfth mirror 53, the dichroic mirror 54, the two-dimensional scanning galvanometer system 55, the scanning lens 56, the field mirror 57, the first Thirteen mirrors 58, objective lens 59, sample stage 60, filter 61, eleventh focusing lens 62, multimode fiber array 63, detector array 64.

The embodiment of the device of the present invention is mainly divided into four parts: an excitation system for generating excitation light, a loss system for generating loss light, a beam splitting system for generating beam arrays, a microscope system for sample imaging and lithography, and a collection of fluorescent signals emitted by the sample. detection system.

Among them, the excitation laser 1, the first single-mode fiber 2, the first collimating lens 3, the first half-wave plate 4, the first quarter-wave plate 5, and the first beam splitter 6 are sequentially arranged in the excitation on the optical axis of the system;

Among them, 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, the first mirror 13 are sequentially arranged on the optical axis of the loss system;

Among them, the second beam splitter to the tenth beam splitter 14-22, the second mirror 23, the first to tenth focusing lenses 24-33, the first to tenth diverging lenses 34-43, the third to tenth A reflector 44-52 and a twelfth reflector 53 are sequentially arranged 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;

Among them, 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;

Using the setup shown in Figure 2, the method used to generate a line of high-throughput ultra-diffraction-limited focal spots is as follows:

1) The excitation laser 1 emits excitation light (the laser with a wavelength of 440 nanometers is used as the excitation light in this embodiment), which is coupled into the first single-mode fiber 2, and then exits from the first single-mode fiber 2 by the first collimating lens 3. Collimated, and then modulated into circularly polarized excitation light through the first half-wave plate 4 and the first quarter-wave plate 5 and then reaches the first beam splitter 6;

2) The lossy laser 7 emits lossy light (the laser with a wavelength of 532 nm is used as the lossy light in this embodiment), which is coupled into the second single-mode fiber 8, and then exits from the second single-mode fiber 8 by the second collimating lens. 9 Collimation, and then pass through the vortex phase plate 10 to become hollow loss light, use the second half wave plate 11 and the second quarter wave plate 12 to convert the hollow loss light into circularly polarized hollow loss light, and then Reflected by the first mirror 13 to the first beam splitter 6;

3) the first beam splitter 6 combines the circularly polarized excitation light and the circularly polarized hollow loss light, and the combined beam is divided into ten beams with equal light intensities by the second beam splitter to the tenth beam splitter, After passing through ten 4f systems composed of the first to tenth focusing lenses 24-33 and the first to tenth diverging lenses 34-43, the beams are condensed, and finally the second reflecting mirror 23 and the third to twelfth mirrors The mirrors 44-53 are reflected to the dichroic mirror 54 to generate a beam array;

4) The 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 beam array and deflects the optical path, and the beam array emitted by the two-dimensional scanning galvanometer system 55 is scanned Distortion is eliminated after lens 56, collimated and beam expanded by field lens 57, reflected by thirteenth mirror 58 to objective lens 59, and finally focused on the sample placed on sample stage 60 by objective lens 59 for stimulated emission Loss generates a high-throughput ultra-diffraction-limited focal spot to scan samples for microscopic imaging or laser direct-write lithography;

5) The high-throughput ultra-diffraction-limited fluorescence emitted by the sample is received by the detector array through the multimode fiber array

The high-throughput ultra-diffraction-limited fluorescence signal emitted by the sample is collected by the objective lens 59, and then reflected on the field lens 57 by the thirteenth reflector 58, and then reaches the scanning galvanometer system 55 through the focusing of the field lens 57 and the collimation of the scanning lens 56. , after de-scanning, it is transmitted to the filter 61 by the dichroic mirror 54 , and the laser light and fluorescence of other wavelengths are filtered out and then focused on the multimode fiber array 63 by the eleventh focusing lens 62 . Finally, the detector array 64 is used to receive in parallel the high-throughput ultra-diffraction-limited fluorescence signals emitted by the sample during the two-dimensional scanning process.

Figure 3 shows the arrangement of the multimode fiber array and the detector array. One hundred multimode fibers form a square multimode fiber array, and one hundred APD detectors form a square detector array.

Figure 4 is a schematic diagram of a high-throughput ultra-diffraction-limited focal spot array in the present invention, wherein Figure A is a schematic diagram of a solid excitation focal spot array, Figure B is a schematic diagram of a hollow loss focal spot array, and Figure C is a generated high-throughput ultra-diffraction-limited focal spot array Schematic diagram of the array. . It can be seen that the half-height full width of the right image is smaller than the left image, which means that the resolution is higher.

The above are only examples of preferred implementations of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. within.

Claims (10)

1. a method for producing high-throughput ultra-diffraction-limited focal spot, is characterized in that, comprises the steps: 1) generate excitation light; 2) modulate the generated loss light into hollow loss light; 3) Combine the excitation light and the hollow loss light into a beam array; 4) Focus the beam array on the sample for stimulated emission loss to generate a high-throughput ultra-diffraction-limited focal spot to scan the sample. 2 . The method for generating a high-throughput ultra-diffraction-limited focal spot according to claim 1 , wherein the excitation light and the hollow loss light are both converted into circularly polarized light and then the sample is scanned. 3 . 3. The method for generating a high-throughput ultra-diffraction-limited focal spot according to claim 1, wherein the beam array is a square array. 4 . The method for generating a high-throughput ultra-diffraction-limited focal spot according to claim 1 , wherein the collection beam array excites the fluorescence emitted by the sample to perform microscopic imaging. 5 . 5. The method for generating a high-throughput ultra-diffraction-limited focal spot as claimed in claim 1, wherein photolithography is performed using a high-throughput ultra-diffraction-limited focal spot focused on the sample by a beam array. 6. A device for producing a high-throughput ultra-diffraction-limited focal spot, comprising: Excitation light source for emitting excitation light; A lossy light source for emitting lossy light; a modulator for modulating lossy light into hollow lossy light; Beam splitting system, used to combine excitation light and hollow loss light into beam array; Microscopy system for SED-generated high-throughput ultra-diffraction-limited focal spot scanning of a sample by focusing an array of beams on a sample. 7. The device for producing high-flux ultra-diffraction-limited focal spot as claimed in claim 6, wherein the excitation light source and the loss light source are divided into multiple groups, and each group comprises an excitation light source and a Loss light source. 8. The device for producing a high-flux ultra-diffraction-limited focal spot as claimed in claim 6 or 7, wherein the beam splitting system is correspondingly arranged on the optical path of each group of excitation light sources and loss light sources, including Set in sequence: a first beam splitter for combining excitation light and hollow loss light; a second beam splitter for dividing the combined beam into a plurality of beams of equal intensity; A 4f system consisting of a pair of lenses for condensing the plurality of beams of equal intensity. 9 . The device for generating a high-throughput ultra-diffraction-limited focal spot according to claim 6 , wherein the modulator is a vortex phase plate. 10 . 10. The device for generating a high-throughput ultra-diffraction-limited focal spot according to claim 6, further comprising a detector array, adapted to the beam array, for collecting the emitted fluorescence for microscopic imaging.

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