CN114895382A - Beam shaping optical element of light sheet fluorescence microscope and light sheet fluorescence microscope - Google Patents
Beam shaping optical element of light sheet fluorescence microscope and light sheet fluorescence microscope Download PDFInfo
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- CN114895382A CN114895382A CN202210432275.7A CN202210432275A CN114895382A CN 114895382 A CN114895382 A CN 114895382A CN 202210432275 A CN202210432275 A CN 202210432275A CN 114895382 A CN114895382 A CN 114895382A
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- 230000003287 optical effect Effects 0.000 title claims abstract description 76
- 238000007493 shaping process Methods 0.000 title claims abstract description 40
- 238000003384 imaging method Methods 0.000 claims abstract description 16
- 239000002184 metal Substances 0.000 claims description 16
- 229910052751 metal Inorganic materials 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 15
- 229920002120 photoresistant polymer Polymers 0.000 claims description 12
- 238000000151 deposition Methods 0.000 claims description 9
- 238000005286 illumination Methods 0.000 claims description 9
- 238000005530 etching Methods 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000005229 chemical vapour deposition Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 238000010894 electron beam technology Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 230000008021 deposition Effects 0.000 claims description 3
- 238000000609 electron-beam lithography Methods 0.000 claims description 3
- 206010034972 Photosensitivity reaction Diseases 0.000 abstract description 3
- 208000007578 phototoxic dermatitis Diseases 0.000 abstract description 3
- 231100000018 phototoxicity Toxicity 0.000 abstract description 3
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000010931 gold Substances 0.000 description 5
- 238000004088 simulation Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- 238000004364 calculation method Methods 0.000 description 1
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- 238000012993 chemical processing Methods 0.000 description 1
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- 238000009826 distribution Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B1/00—Optical elements characterised by the material of which they are made; Optical coatings for optical elements
- G02B1/002—Optical elements characterised by the material of which they are made; Optical coatings for optical elements made of materials engineered to provide properties not available in nature, e.g. metamaterials
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/09—Beam shaping, e.g. changing the cross-sectional area, not otherwise provided for
- G02B27/0938—Using specific optical elements
Abstract
The invention discloses a beam shaping optical element of an optical sheet fluorescence microscope and an optical sheet fluorescence microscope, wherein the optical element is provided with a spiral-structured super-structure surface, and the spiral-structured super-structure surface has the following dimensional characteristics: the terminal radius r of the helical structure is determined as follows:wherein λ is spp Is the resonant wavelength of the metamaterial surface plasmon element,angle of rotation of helical structure, r 0 Is the initial radius of the spiral structure. The invention provides a beam shaping optical element of an optical sheet fluorescence microscopeThe optical wavefront regulation and control capability of the sub-wavelength spatial resolution is realized, the optical wavefront can be precisely shaped, and the non-diffraction light beam with the light spot size within the sub-wavelength range is obtained, so that the imaging performance of the medical light sheet microscope is improved, the phototoxicity to an imaging target object is reduced, and the application requirement of noninvasive extraction of living cell information in life science can be better met.
Description
Technical Field
The invention relates to the technical field of optical imaging, in particular to a beam shaping optical element of an optical sheet fluorescence microscope and the optical sheet fluorescence microscope.
Background
In life science research, it is a long-standing pursuit to non-invasively extract as much spatiotemporal information as possible from living cells. This requires that the imaging system achieve a larger field of view, higher resolution, and higher speed three-dimensional images without affecting the biological activity. Optical microscopy imaging technology has evolved rapidly, from traditional wide field fluorescence microscopy to the most advanced light sheet microscopy. The Light Sheet Fluorescence Microscope (LSFM) is characterized in that the lighting mode of the exciting light is that the lighting optical axis is vertical to the detection optical axis, the lighting light adopts a thin 'light sheet' parallel to the imaging surface, only the sample of the focal plane is lighted, the sample part above and below the sample part is not affected, and the light sheet fluorescence microscope has a natural optical slicing function. The principle of the light sheet fluorescence microscope is shown in FIG. 1. The key core technology of the light sheet fluorescence microscope is the generation of a light sheet, and the simplest method is to introduce a cylindrical lens in a light path. The Gaussian beam passes through the cylindrical lens, and the focused light waist omega 0 λ/(π × NA), which is the thickness of the optical sheet, and the confocal parameter b is 2 ω 0 and/NA is the effective visual field length. In most cases, the numerical aperture NA of the imaging objective is low, the light sheet thickness is smaller than the depth of field, and the axial resolution of the system is determined by the light sheet thickness. The now common light sheet system is a digital scanning light sheet microscope that generates a virtual light sheet by scanning a focused light beam perpendicular to the detection axis in a single direction.
Light sheet microscopy technology has evolved around light sheets. By precisely manipulating the light sheet, the performance of LSFMs is improved: higher resolution, larger field of view, better optical sectioning, faster imaging speed, and reduced photobleaching and phototoxicity. The existing common technical approach for improving the performance of a light sheet microscope (light sheet performance) is to adjust the amplitude and phase of an optical wavefront at the pupil function of a microscope objective lens, and adjust the amplitude and phase of the optical wavefront by introducing a passive optical element (such as an amplitude mask, a phase mask and an axicon) or an active wavefront shaping device (such as a spatial light modulator and a deformable mirror), so as to complete beam shaping and realize a required light sheet. The disadvantages of these techniques are:
1) the traditional passive optical element is adopted for beam shaping, so that incident light cannot be regulated and controlled on a sub-wavelength scale, and the improvement of resolution is limited;
2) the active wavefront shaping device is adopted for beam shaping, the adjustment technical requirement is high, the stability is low, and the cost is high.
Therefore, there is a need to provide a more reliable solution.
Disclosure of Invention
The present invention provides a beam shaping optical element of an optical sheet fluorescence microscope and an optical sheet fluorescence microscope, which aim at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a light sheet fluorescence microscope beam shaping optical element, the optical element having a helical structured nanostructured surface having the following dimensional characteristics:
the terminal radius r of the helical structure is determined as follows:
wherein λ is spp Is the resonant wavelength of the metamaterial surface plasmon element,angle of rotation of helical structure, r 0 The initial radius of the spiral structure; lambda [ alpha ] 0 Is the wavelength of the incident wave,. epsilon d Is the dielectric constant of air, epsilon' m Is the dielectric constant of the metamaterial surface material.
Preferably, the height of the nanostructured surface of the helical structure is 20-30 nm.
Preferably, the width of the helical structure is 150-250 nm.
Preferably, the helical structure has a rotation angle of 1080 °, i.e. 3 turns.
Preferably, the width of the helical structure matched to an incident wavelength of 800nm is 200 nm.
Preferably, the preparation method comprises the following steps:
1) establishing a three-dimensional model of the beam shaping optical element of the optical sheet fluorescence microscope according to the size characteristics;
2) providing a clean substrate;
3) uniformly coating photoresist on the surface of the substrate in a rotary coating mode;
4) depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode for conducting current of electron beam etching;
5) carrying out electron beam lithography exposure on the photoresist according to the three-dimensional model established in the step 1), and forming an exposure gap by etching the metal Cr film;
6) sequentially depositing metal Cr and metal Au, wherein the deposition thickness of the metal Cr is 1-2 nm;
7) and stripping the photoresist to obtain the beam shaping optical element of the optical sheet fluorescence microscope.
Preferably, acetone, isopropanol and O are sequentially used in the step 2) 2 And cleaning the substrate material by using the plasma to obtain a clean substrate, wherein the substrate is a glass substrate.
The invention also provides a light sheet fluorescence microscope, which comprises the beam shaping optical element of the light sheet fluorescence microscope.
Preferably, the light sheet fluorescence microscope further comprises an illumination light source, an illumination objective lens and a detection objective lens, and the light sheet fluorescence microscope beam shaping optical element is arranged between the illumination objective lens and the imaging sample.
Preferably, the distance between the light sheet fluorescence microscope beam shaping optical element and the imaging position is not more than 1 cm.
The invention has the beneficial effects that:
the optical sheet fluorescence microscope beam shaping optical element provided by the invention has the optical wavefront regulation and control capability of sub-wavelength spatial resolution, can accurately shape the wavefront of light to obtain the non-diffraction light beam with the light spot size within the sub-wavelength, thereby improving the imaging performance of a medical optical sheet microscope, reducing the phototoxicity on an imaging target object, and better meeting the application requirement of noninvasive extraction of living cell information in life science.
Drawings
FIG. 1 is a schematic diagram of a light sheet fluorescence microscope;
FIG. 2 is a scanning electron micrograph of a nanostructured surface of a helical structure in example 1 of the present invention;
fig. 3 is a simulation diagram of the beam shaping of the super-structured surface of the helical structure in embodiment 1 of the present invention;
FIG. 4 is a flow chart of the preparation of beam shaping optical element of an optical sheet fluorescence microscope in example 1 of the present invention;
fig. 5 is a schematic structural view of a light sheet fluorescence microscope in embodiment 2 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
A beam shaping optical element of an optical sheet fluorescence microscope is provided with a spiral-structured super-structure surface, the shape and the size of the super-structure surface are determined by combining light source parameters of the optical sheet microscope and theoretical calculation according to a geometric phase and a surface plasmon resonance mechanism, and specifically, the spiral-structured super-structure surface has the following size characteristics:
the terminal radius r of the helical structure is determined as follows:
wherein λ is spp Is the resonant wavelength of the metamaterial surface plasmon element,angle of rotation of helical structure, r 0 The initial radius of the spiral structure; lambda [ alpha ] 0 Is the wavelength of the incident wave,. epsilon d Is the dielectric constant of air, epsilon' m Is the dielectric constant of the metamaterial surface material.
In a preferred embodiment, the height (H) of the nanostructured surface of the helical structure is 20-30nm, the width (W) of the helical structure is 150-250nm, and the rotation angle of the helical structure is 1080 °, i.e. 3 turns.
In a preferred embodiment, the width of the spiral structure matched with the incident wavelength of 800nm is 200nm, and the specific structure size is shown in fig. 2, wherein, in the embodiment, the surface material of the metamaterial is gold, and the plasmon resonance wavelength of the metal gold on the surface of the glass substrate is 787.5 nm.
In this embodiment, a three-dimensional physical model is established for the design of the above-described super-structured surface, and a simulation grid is set reasonably. By means of Maxwell equation solution and Fourier mode, aiming at strict vector simulation of the micro-structure at the focus, the change of energy, phase and polarization distribution of a focused light beam after the interaction with the super-structure surface is simulated. The simulation result is shown in fig. 3, which shows that the light beam is focused at the center of the spiral micro-nano structure, and the diameter of the light spot is smaller than the wavelength.
In a preferred embodiment, the beam shaping optical element of the optical sheet fluorescence microscope is prepared by using main processes such as Chemical Vapor Deposition (CVD), electron beam etching and the like, and matching with a common chemical processing technical means in an ultraclean room, and with reference to fig. 4, the beam shaping optical element specifically includes the following steps:
1) establishing a three-dimensional model of the beam shaping optical element of the optical sheet fluorescence microscope according to the size characteristics;
2) using acetone, isopropanol and O in sequence 2 Plasma cleaning the glass substrate;
3) uniformly coating photoresist on the surface of the substrate in a rotary coating mode;
4) depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode for conducting current of electron beam etching;
5) carrying out electron beam lithography exposure on the photoresist according to the three-dimensional model established in the step 1), and forming an exposure gap by etching the metal Cr film;
6) sequentially depositing metal Cr and metal Au; in a preferred embodiment, the deposition thickness of the metal Cr is 1-2 nm; in the process, the Au deposited directly can easily fall off, so that the Cr is adopted as an adhesion layer and a very thin adhesion layer is adopted;
7) and stripping the photoresist to obtain the beam shaping optical element of the optical sheet fluorescence microscope.
Example 2
This embodiment provides an optical sheet fluorescence microscope, which includes the beam shaping optical element of the optical sheet fluorescence microscope in embodiment 1, and necessary components such as an illumination light source, an illumination objective lens, and a detection objective lens, wherein the beam shaping optical element of the optical sheet fluorescence microscope is vertically disposed between the illumination objective lens and an imaging sample and is as close as possible to an imaging position according to the requirement of its working state, and in a preferred embodiment, the distance between the beam shaping optical element of the optical sheet fluorescence microscope and the imaging position is not more than 1 cm.
According to the invention, by designing a proper figure and size of the micro-nano ultrastructural surface, a light beam can be shaped by the micro-nano ultrastructural surface optical element to obtain a non-diffraction light beam with a light spot size within a sub-wavelength.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A beam shaping optical element for an optical sheet fluorescence microscope, the optical element having a helical textured surface with the following dimensional characteristics:
the terminal radius r of the helical structure is determined as follows:
wherein λ is spp Is the resonant wavelength of the metamaterial surface plasmon element,angle of rotation of helical structure, r 0 The initial radius of the spiral structure; lambda [ alpha ] 0 Is the wavelength of the incident wave,. epsilon d Is the dielectric constant of air, epsilon' m Is the dielectric constant of the metamaterial surface material.
2. The light sheet fluorescence microscope beam shaping optical element of claim 1, wherein the height of the spiral structured super-structured surface is 20-30 nm.
3. The optical sheet fluorescence microscope beam shaping optical element according to claim 2, wherein the width of the spiral structure is 150 nm and 250 nm.
4. The optical sheet fluorescence microscope beam shaping optical element according to claim 3, wherein the rotation angle of the spiral structure is 360-1080 °, i.e. 1-3 turns.
5. The optical sheet fluorescence microscope beam shaping optical element according to claim 4, wherein the width of the helical structure used is 200nm corresponding to an incident wavelength of 800 nm.
6. The light sheet fluorescence microscope beam shaping optical element according to claim 1, characterized in that it is prepared by the following method:
1) establishing a three-dimensional model of the beam shaping optical element of the optical sheet fluorescence microscope according to the size characteristics;
2) providing a clean substrate;
3) uniformly coating photoresist on the surface of the substrate in a rotary coating mode;
4) depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode for conducting current of electron beam etching;
5) carrying out electron beam lithography exposure on the photoresist according to the three-dimensional model established in the step 1), and forming an exposure gap by etching the metal Cr film;
6) sequentially depositing metal Cr and metal Au, wherein the deposition thickness of the metal Cr is 1-2 nm;
7) and stripping the photoresist to obtain the beam shaping optical element of the optical sheet fluorescence microscope.
7. The light sheet fluorescence microscope beam shaping optical element according to claim 6, wherein acetone, isopropanol and O are sequentially used in the step 2) 2 And cleaning the substrate material by using the plasma to obtain a clean substrate, wherein the substrate is a glass substrate.
8. A light sheet fluorescence microscope comprising the light sheet fluorescence microscope beam shaping optical element of any of claims 1-7.
9. The light sheet fluorescence microscope of claim 8, further comprising an illumination source, an illumination objective, and a detection objective, the light sheet fluorescence microscope beam shaping optics disposed between the illumination objective and the imaging sample.
10. The light sheet fluorescence microscope of claim 8, wherein the distance between the light sheet fluorescence microscope beam shaping optical element and an imaging location is no greater than 1 cm.
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