CN114895382B - 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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- CN114895382B CN114895382B CN202210432275.7A CN202210432275A CN114895382B CN 114895382 B CN114895382 B CN 114895382B CN 202210432275 A CN202210432275 A CN 202210432275A CN 114895382 B CN114895382 B CN 114895382B
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- 230000003287 optical effect Effects 0.000 title claims abstract description 47
- 238000007493 shaping process Methods 0.000 title claims abstract description 33
- 238000003384 imaging method Methods 0.000 claims abstract description 18
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 claims abstract description 5
- 239000002184 metal Substances 0.000 claims description 14
- 229910052751 metal Inorganic materials 0.000 claims description 14
- 239000000758 substrate Substances 0.000 claims description 13
- 238000005286 illumination Methods 0.000 claims description 12
- 229920002120 photoresistant polymer Polymers 0.000 claims description 12
- 238000000151 deposition Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 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
- 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
- 238000010894 electron beam technology Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 238000000609 electron-beam lithography Methods 0.000 claims description 3
- 238000007747 plating Methods 0.000 claims description 3
- 230000008021 deposition Effects 0.000 claims description 2
- 206010034972 Photosensitivity reaction Diseases 0.000 abstract description 3
- 238000000605 extraction Methods 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
- 239000010931 gold Substances 0.000 description 4
- 238000004088 simulation Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process 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
- 239000002086 nanomaterial Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012993 chemical processing Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000005284 excitation Effects 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
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012634 optical imaging Methods 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
Classifications
-
- 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 a light sheet fluorescence microscope and the light sheet fluorescence microscope, wherein the optical element is provided with an ultra-structured surface with a spiral structure, and the ultra-structured surface with the spiral structure has the following dimensional characteristics: the radius r of the spiral structure terminal is determined by:wherein lambda is spp Is the super-structured surface plasmon resonance wavelength,is the rotation angle of a spiral structure, r 0 Is the initial radius of the spiral structure. The beam shaping optical element of the light sheet fluorescence microscope 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 non-diffracted light beams with the light spot size within the sub-wavelength, thereby improving the imaging performance of the medical light sheet microscope, reducing the phototoxicity to imaging targets, and better meeting the application requirements of noninvasive extraction of living cell information in life science.
Description
Technical Field
The invention relates to the technical field of optical imaging, in particular to a beam shaping optical element of a light sheet fluorescence microscope and the light sheet fluorescence microscope.
Background
In life sciences research, noninvasive extraction of as much spatial and temporal information as possible from living cells has long been sought. This requires the imaging system to achieve a larger field of view, and more, without affecting biological activityHigh resolution, higher speed three-dimensional images. Optical microscopy imaging techniques have evolved rapidly from traditional wide-field fluorescence microscopy imaging to the most advanced light sheet microscopy imaging. The Light Sheet Fluorescence Microscope (LSFM) is characterized by that the illumination mode of excitation light, i.e. the illumination optical axis and detection optical axis are mutually perpendicular, and the illumination light adopts a thin "light sheet" parallel to imaging surface, only the sample of focal plane is illuminated, and its upper and lower sample portions are not affected, so that it has 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 the light sheet, and the simplest method is to introduce a cylindrical lens in the light path. Gaussian beam passes through cylindrical lens, and focused light waist omega 0 Let λ/(pi×na), i.e. the thickness of the light sheet, confocal parameter b=2ω 0 NA, the effective field length. In most cases, the numerical aperture NA of the imaging objective is low, the thickness of the light sheet is smaller than the depth of field, and the axial resolution of the system is determined by the thickness of the light sheet. A common light sheet system today generates a virtual light sheet by scanning a focused light beam perpendicular to the detection axis in a single direction, known as a digital scanning light sheet microscope.
Development of light sheet microscopy has been around the light sheet. Through accurate operation light piece, improve the performance of LSFMs: higher resolution, larger field of view, better optical sectioning, faster imaging speeds, and reduced photobleaching and phototoxicity. The conventional 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, 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 the 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 wave front shaping device is adopted for shaping the light beam, the technical requirement for adjustment 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 invention aims to solve the technical problem of providing a beam shaping optical element of a light sheet fluorescence microscope and the light sheet fluorescence microscope aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: an optical element for beam shaping of an optical sheet fluorescence microscope, the optical element having a super-structured surface of a spiral structure, the super-structured surface of the spiral structure having the following dimensional characteristics:
the radius r of the spiral structure terminal is determined by:
wherein lambda is spp Is the super-structured surface plasmon resonance wavelength,is the rotation angle of a spiral structure, r 0 Is the initial radius of the spiral structure; lambda (lambda) 0 Epsilon for the wavelength of the incident wave d Is air dielectric constant, epsilon' m Is the dielectric constant of the super-structured surface material.
Preferably, the height of the superstructural surface of the spiral structure is 20-30nm.
Preferably, the width of the helical structure is 150-250nm.
Preferably, the helical structure has a rotational angle of 1080 °, i.e. 3 turns.
Preferably, the width of the helix matching the incident wavelength is 200nm.
Preferably, it is prepared by the following method:
1) Establishing a three-dimensional model of the beam shaping optical element of the light 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 film plating mode;
4) Depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode, and conducting current for electron beam etching;
5) Carrying out electron beam lithography exposure photoresist according to the three-dimensional model established in the step 1), and forming an exposure notch by etching the metal Cr film;
6) Sequentially depositing metal Cr and metal Au, wherein the deposition thickness of the metal Cr is 1-2nm;
7) And stripping the photoresist to obtain the beam shaping optical element of the light sheet fluorescence microscope.
Preferably, acetone, isopropanol and O are used in this order in step 2) 2 The substrate material is plasma cleaned to obtain a clean substrate, which is a glass substrate.
The invention also provides an optical sheet fluorescence microscope comprising the optical sheet fluorescence microscope beam shaping optical element.
Preferably, the light sheet fluorescence microscope further comprises an illumination light source, an illumination objective and a detection objective, and the light sheet fluorescence microscope beam shaping optical element is arranged between the illumination objective and the imaging sample.
Preferably, the distance between the beam shaping optical element of the light sheet fluorescence microscope and the imaging location is not more than 1cm.
The beneficial effects of the invention are as follows:
the beam shaping optical element of the light sheet fluorescence microscope 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 non-diffracted light beams with the light spot size within the sub-wavelength, thereby improving the imaging performance of the medical light sheet microscope, reducing the phototoxicity to imaging targets, and better meeting the application requirements 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 super-structured surface of a spiral structure in example 1 of the present invention;
FIG. 3 is a simulation diagram of the super-structured surface beam shaping of the spiral structure in example 1 of the present invention;
FIG. 4 is a flow chart of the preparation of beam shaping optics for a light sheet fluorescence microscope in example 1 of the present invention;
fig. 5 is a schematic structural diagram of a light sheet fluorescence microscope in example 2 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring 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
The optical element is provided with a super-structured surface with a spiral structure, the shape and the size of the super-structured surface are determined by theoretical calculation according to geometric phase and surface plasmon resonance mechanism and combining with light source parameters of a light sheet microscope, and specifically, the super-structured surface with the spiral structure has the following size characteristics:
the radius r of the spiral structure terminal is determined by:
wherein lambda is spp Is the super-structured surface plasmon resonance wavelength,is the rotation angle of a spiral structure, r 0 Is the initial radius of the spiral structure; lambda (lambda) 0 Epsilon for the wavelength of the incident wave d Is air dielectric constant, epsilon' m Is the dielectric constant of the super-structured surface material.
In a preferred embodiment, the height (H) of the superstructural surface of the spiral structure is 20-30nm, the width (W) of the spiral structure is 150-250nm, and the rotation angle of the spiral 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 super-structure surface material is gold, and the resonance wavelength of the plasmon element for metallic gold on the surface of the glass substrate is 787.5nm.
In this embodiment, a three-dimensional physical model is built for the design of the above super-structured surface, and a simulation grid is reasonably set. The change of energy, phase and polarization distribution of the focused light beam after the interaction of the focused light beam and the super-structured surface is simulated by a Maxwell equation method and a Fourier mode method aiming at the strict vector simulation of the micro-nano structure at the focus. The simulation results are shown in fig. 3, which shows that the beam is focused at the center of the spiral micro-nano structure and the spot diameter is smaller than the wavelength.
In a preferred embodiment, the beam shaping optical element of the light sheet fluorescence microscope is prepared by adopting main processes such as Chemical Vapor Deposition (CVD) and electron beam etching, and is matched with a common chemical processing technology means of an ultra clean room, and referring to fig. 4, the beam shaping optical element specifically comprises the following steps:
1) Establishing a three-dimensional model of the beam shaping optical element of the light sheet fluorescence microscope according to the size characteristics;
2) Sequentially using acetone, isopropanol and O 2 Plasma cleaning the glass substrate;
3) Uniformly coating photoresist on the surface of the substrate in a rotary film plating mode;
4) Depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode, and conducting current for electron beam etching;
5) Carrying out electron beam lithography exposure photoresist according to the three-dimensional model established in the step 1), and forming an exposure notch by etching the metal Cr film;
6) Sequentially depositing metal Cr and metal Au; in a preferred embodiment, the metallic Cr is deposited to a thickness of 1-2nm; wherein, au can be easily dropped when being directly deposited on the process, so 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 light sheet fluorescence microscope.
Example 2
The present embodiment provides an optical sheet fluorescence microscope, which includes the optical sheet fluorescence microscope beam shaping optical element of embodiment 1, and necessary components such as an illumination light source, an illumination objective, a detection objective, etc., according to the working state requirements, the optical sheet fluorescence microscope beam shaping optical element is vertically disposed between the illumination objective and the imaging sample and is as close to the imaging position as possible, and in a preferred embodiment, the distance between the optical sheet fluorescence microscope beam shaping optical element and the imaging position is not greater than 1cm.
The invention can obtain the non-diffraction light beam with the light spot size within the sub-wavelength after the light beam is shaped by the micro-nano super-structured surface optical element by designing the proper pattern and the size of the micro-nano super-structured surface.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (2)
1. The light sheet fluorescence microscope is characterized by comprising a light sheet fluorescence microscope beam shaping optical element, an illumination light source, an illumination objective lens and a detection objective lens, wherein the light sheet fluorescence microscope beam shaping optical element is arranged between the illumination objective lens and an imaging sample;
the distance between the beam shaping optical element of the light sheet fluorescence microscope and the imaging position is not more than 1cm;
the beam shaping optical element of the light sheet fluorescent microscope has an ultra-structured surface with a spiral structure, and the ultra-structured surface with the following dimensional characteristics:
the radius r of the spiral structure terminal is determined by:
wherein lambda is spp Is the super-structured surface plasmon resonance wavelength,is the rotation angle of a spiral structure, r 0 Is the initial radius of the spiral structure; lambda (lambda) 0 Epsilon for the wavelength of the incident wave d Is air dielectric constant, epsilon' m Is the dielectric constant of the super-structured surface material;
the height of the super-structured surface of the spiral structure is 20-30nm, and the width of the spiral structure is 150-250nm; the rotation angle of the spiral structure is 360-1080 degrees, namely 1-3 circles;
the width of the adopted spiral structure is 200nm corresponding to the incident wavelength of 800 nm;
the light sheet fluorescence microscope is prepared by the following method:
1) Establishing a three-dimensional model of the beam shaping optical element of the light 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 film plating mode;
4) Depositing metal Cr on the surface of the photoresist in a chemical vapor deposition mode, and conducting current for electron beam etching;
5) Carrying out electron beam lithography exposure photoresist according to the three-dimensional model established in the step 1), and forming an exposure notch by etching the metal Cr film;
6) Sequentially depositing metal Cr and metal Au, wherein the deposition thickness of the metal Cr is 1-2nm;
7) And stripping the photoresist to obtain the beam shaping optical element of the light sheet fluorescence microscope.
2. The light sheet fluorescence microscope of claim 1, wherein acetone, isopropanol and O are sequentially used in step 2) 2 The substrate material is plasma cleaned to obtain a clean substrate, which is a glass substrate.
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| CN202210432275.7A CN114895382B (en) | 2022-04-22 | 2022-04-22 | Beam shaping optical element of light sheet fluorescence microscope and light sheet fluorescence microscope |
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| CN202210432275.7A CN114895382B (en) | 2022-04-22 | 2022-04-22 | Beam shaping optical element of light sheet fluorescence microscope and light sheet fluorescence microscope |
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