CN111856737B - Two-photon light field calculation microscope objective - Google Patents
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- CN111856737B CN111856737B CN202010667790.4A CN202010667790A CN111856737B CN 111856737 B CN111856737 B CN 111856737B CN 202010667790 A CN202010667790 A CN 202010667790A CN 111856737 B CN111856737 B CN 111856737B
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- 238000004364 calculation method Methods 0.000 title claims abstract description 24
- 230000003287 optical effect Effects 0.000 claims abstract description 29
- 238000003384 imaging method Methods 0.000 claims abstract description 21
- 238000013461 design Methods 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000005284 excitation Effects 0.000 claims abstract description 13
- 230000004075 alteration Effects 0.000 claims description 8
- 239000010627 cedar oil Substances 0.000 claims description 6
- 230000005499 meniscus Effects 0.000 claims description 6
- 206010010071 Coma Diseases 0.000 claims description 2
- 238000013459 approach Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000010586 diagram Methods 0.000 description 9
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- 239000000463 material Substances 0.000 description 4
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- 230000008569 process Effects 0.000 description 4
- 238000004026 adhesive bonding Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000012938 design process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- G—PHYSICS
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- G02B21/00—Microscopes
- G02B21/02—Objectives
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0028—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders specially adapted for specific applications, e.g. for endoscopes, ophthalmoscopes, attachments to conventional microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0072—Optical details of the image generation details concerning resolution or correction, including general design of CSOM objectives
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Abstract
The invention discloses a two-photon light field calculation microscope objective which adopts a reverse design idea for matching with a scanning excitation system and comprises a beam expanding lens group BE, a dichroic mirror D, a water mirror O and a medium W which are sequentially arranged along an optical axis; the objective lens is of an asymmetrical structure of an excitation aperture and a collection aperture, adopts a split aperture design, excites fluorescence with a small numerical aperture, collects energy with a large numerical aperture, and greatly reduces optical design and manufacturing pressure while achieving near diffraction limit resolution; the beam expander set BE comprises a focusing set G1 and a collimating set G2 and is used for focusing incident parallel light once and then expanding and collimating the incident parallel light, the dichroic mirror D is used for dividing exciting light and excited fluorescent light into respective light paths, the water mirror O comprises a similar beam expander set G3 and a focusing set G4 and is used for expanding the incident light and then focusing and imaging the expanded incident light, the imaging quality is improved, and the medium W is used for increasing the numerical aperture; the resulting objective lens has a higher resolution than a conventional objective lens of the same field of view.
Description
Technical Field
The invention belongs to the field of optics, and particularly relates to a two-photon light field calculation microscope objective.
Background
Further breakthroughs in tumor therapy require an understanding of the immune processes occurring in the cellular microenvironment from the cellular population level, which requires imaging instruments capable of high resolution three-dimensional imaging with a large field of view. The field-resolution ratio of the existing microscope is limited due to the limited space bandwidth product, and the field range is difficult to improve on the premise of pursuing high resolution; in two-photon excitation, the aim of improving the resolution can be achieved by increasing the numerical aperture of an objective lens, but the aim is achieved at the cost of losing the penetration depth; in addition, the traditional 'excitation-imaging' common objective mechanism needs a small numerical aperture to improve the penetration depth, but needs a large numerical aperture to improve the imaging resolution, and has an irreconcilable contradiction. In a word, the existing objective lens has mutual restriction on large visual field, penetration depth and high resolution index, cannot simultaneously take into account, cannot meet the research requirement of life science, and needs to be improved and broken through urgently.
Disclosure of Invention
The embodiment of the invention aims to provide a two-photon light field calculation microscope objective lens to solve the problem that the existing objective lens has mutual restriction on large view field, penetration depth and high resolution index and cannot simultaneously give consideration to the problem.
In order to achieve the purpose, the invention adopts the following technical scheme: a two-photon light field calculation microscope objective is of an asymmetrical structure of an excitation aperture and a collection aperture, adopts a split aperture design, excites fluorescence with a small numerical aperture, and collects energy with a large numerical aperture; the two-photon light field calculation microscope objective comprises a beam expander group BE, a dichroic mirror D, a water mirror O and a medium W which are sequentially arranged along an optical axis; the beam expander set BE comprises a focusing set G1 and a collimating set G2 and is used for focusing incident parallel light once and then expanding and collimating the incident parallel light, the dichroic mirror D is used for dividing exciting light and excited fluorescent light into respective light paths, the water mirror O comprises a similar beam expander set G3 and a focusing set G4 and is used for expanding the incident light and then focusing and imaging the expanded incident light, the imaging quality is improved, and the medium W is used for increasing the numerical aperture.
Furthermore, the numerical aperture of the two-photon light field calculation microscope objective is 0.3-1.0, the object space view field diameter is 1mm-10mm, the object space transverse resolution is 0.3 μm-2 μm, the exciting light wavelength range is 400nm-1000nm, the fluorescence wavelength range is 460nm-700nm, and the axial resolution is 1 μm-10 μm.
Further, the focusing lens group G1 includes a first convex-concave lens L1, a first biconvex lens L2, a second biconvex lens L3, and a second convex-concave lens L4, which are arranged in order in the optical axis direction.
Further, the collimator lens group G2 includes a first double concave lens L5, a first meniscus lens L6, and a third double convex lens L7 arranged in this order in the optical axis direction.
Further, the beam expander-like group G3 includes a fourth biconvex lens L8, a second biconcave lens L9, a third biconcave lens L10, a second meniscus lens L11, and a fifth biconvex lens L12, which are arranged in this order in the optical axis direction.
Further, the focusing mirror group G4 includes a first convex-flat lens L13, a first plano-concave lens L14, a sixth biconvex lens L15, and a second convex-flat lens L16, which are arranged in this order in the optical axis direction.
Further, the beam expanding mirror group BE can effectively adjust the diameter and the divergence angle of the light beam, so that the smaller the divergence angle is, the better the light beam emitted by the beam expanding mirror group BE meets the condition of matching the size of the machinable dichroic mirror D.
Further, the beam expander group BE can BE combined with the water mirror O to correct various aberrations mainly including spherical aberration and coma aberration, so that the resolution of each field approaches to the diffraction limit.
Further, the medium W is water, cedar oil or cedar oil.
According to the technical scheme, the objective lens with the asymmetrical structure of the excitation aperture and the collection aperture can be obtained by optimally designing the position and the parameters of each lens group, the objective lens can excite fluorescence with small numerical aperture, collects energy with large numerical aperture, greatly reduces optical design and manufacturing pressure while achieving near-diffraction limit resolution of a full-field in a working waveband, and has higher imaging resolution compared with the traditional objective lens with the same field of view; in addition, the sub-aperture design improves robustness to scattering and helps to increase the system signal-to-noise ratio.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is an optical system diagram of a two-photon optical field computing microscope objective according to a preferred embodiment of the present invention;
FIG. 2 is a diagram of the modulation transfer function of a two-photon optical field calculation microscope objective according to a preferred embodiment of the present invention;
FIG. 3 is a schematic diagram of a two-photon optical field calculation microscope objective lens according to a preferred embodiment of the present invention, in which different shapes represent different wavelengths, + represents 910nm, a square represents 920nm, and a triangle represents 930 nm;
FIG. 4 is a field curvature and distortion diagram of a two-photon light field calculation microscope objective lens according to a preferred embodiment of the present invention, wherein (a) is a field curvature diagram and (b) is a distortion diagram;
FIGS. 5-13 are diagrams of sub-aperture optical systems with representative fields of view and scan angles;
FIGS. 14-22 are graphs of modulation transfer functions corresponding to the sub-apertures shown in FIGS. 5-13;
fig. 23 to 31 are dot-sequence diagrams corresponding to the sub-apertures shown in fig. 5 to 13, and the wavelength explanation is the same as that of fig. 3.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
Exemplary embodiments according to the present application will now be described in more detail with reference to the accompanying drawings. These exemplary embodiments may, however, be embodied in many different forms and should not be construed as limited to only the embodiments set forth herein. It is to be understood that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art, in the drawings, it is possible to enlarge the thicknesses of layers and regions for clarity, and the same devices are denoted by the same reference numerals, and thus the description thereof will be omitted.
Example 1:
as shown in fig. 1, the present embodiment provides a two-photon light field calculation microscope objective, which is an asymmetric structure of an excitation aperture and a collection aperture, adopts a split aperture design to excite fluorescence with a small numerical aperture, and collects energy with a large numerical aperture, thereby greatly reducing optical design and manufacturing pressure while achieving a near diffraction limit resolution; the two-photon light field calculation microscope objective comprises a beam expander group BE, a dichroic mirror D, a water mirror O and a medium W which are sequentially arranged along an optical axis; the beam expander set BE comprises a focusing set G1 and a collimating set G2 and is used for focusing incident parallel light once and then expanding and collimating the incident parallel light, the dichroic mirror D is used for dividing exciting light and excited fluorescent light into respective light paths, the water mirror O comprises a similar beam expander set G3 and a focusing set G4 and is used for expanding incident light and then focusing and imaging the expanded incident light, the imaging quality is improved, and the medium W is used for increasing the numerical aperture.
Furthermore, the numerical aperture of the two-photon light field calculation microscope objective is 0.3-1.0, the object space view field diameter is 1mm-10mm, the object space transverse resolution is 0.3 μm-2 μm, the exciting light wavelength range is 400nm-1000nm, the fluorescence wavelength range is 460nm-700nm, and the axial resolution is 1 μm-10 μm. The embodiment is described by using one example, and those skilled in the art can know how to design the remaining value ranges by using the following example, which is specifically as follows: the numerical aperture of the objective lens is set to be 1.0, the diameter of an object space view field is set to be 4mm, the transverse resolution of the object space is set to be 0.5 mu m, the excitation wavelength range is 910nm-930nm, the fluorescence wavelength range is 460nm-700nm, and the axial resolution is set to be 1.5 mu m. The initial structure of the objective lens is given in the form of a complete aperture (fig. 1), each sub-aperture is optimally designed by using a multiple structure in the design process, and partial optical system diagrams and imaging effect analysis with representative sub-apertures are shown in fig. 5-fig. 31. In the using process of the microscope, femtosecond laser with the wavelength of 910nm to 930nm is adopted to scan samples at different positions and angles with the numerical aperture of 0.2, the scanning area is 3mm multiplied by 3mm each time, the scanning angle of the same position of the sample at different exit pupil positions of the laser is-48 degrees to 48 degrees, and for each excited fluorescence point, fluorescence with the wavelength of 460nm to 700nm is collected by a high-energy collecting system with the NA being 1.0. The center of the system is provided with a dichroic mirror, scanning femtosecond laser enters from the dichroic mirror, and a light path is divided into two parts by taking the dichroic mirror as a boundary; and the exit end of the dichroic mirror is a high-energy acquisition system. Firstly, adjusting light paths of an emergent end and an incident end of a dichroic mirror to enable principal rays to be parallel; secondly, the scanning excitation system is designed and optimized to reach the limit resolution. The energy collection detector is large in size, so that the requirement on the size of a light spot of a high-energy collection system is low, and the system design is relatively easy to realize. In a word, the limit resolution scanning is realized by a small numerical aperture, the energy collection is realized by a large numerical aperture instead of imaging, the difficulty and the cost of optical design are greatly reduced, and the penetration depth is considered while the field-resolution ratio is broken through.
Specifically, the focusing lens group G1 adopts a structure of a group of double cemented lenses, a double convex lens and a thick meniscus lens to primarily converge light, and has a large focal power, and includes a first convex-concave lens L1, a first double convex lens L2, a second double convex lens L3 and a second convex-concave lens L4, which are sequentially arranged in the optical axis direction.
Specifically, the collimating lens group G2 uses a double concave thin lens, a meniscus thick lens and a double convex thin lens to expand and collimate the primary convergent light beam, and forms a quasi-symmetric structure with the focusing lens group G1, and completes adjustment of the incident light beam diameter and the divergence angle together, including a first double concave lens L5, a first meniscus lens L6 and a third double convex lens L7 which are sequentially arranged along the optical axis direction.
Specifically, the beam expander-like lens group G3 compensates aberration introduced by the dichroic mirror using a lens group composed of two groups of glass pairs with large abbe numbers difference and a double convex thick lens, and further expands and adjusts the beam to improve the final imaging quality, and includes a fourth double convex lens L8, a second double concave lens L9, a third double concave lens L10, a second concave-convex lens L11 and a fifth double convex lens L12, which are sequentially arranged along the optical axis direction.
Specifically, the focusing lens group G4 adopts a structure of combining a plurality of thick lenses and positive and negative lenses to finally focus light, also bears a large focal power, corrects various aberrations introduced by the lens groups before, and balances and improves the resolution of each field of view, and includes a first convex-flat lens L13, a first plano-concave lens L14, a sixth biconvex lens L15, and a second convex-flat lens L16, which are sequentially arranged along the optical axis direction.
Specifically, the medium W can be water, cedar oil and the like, and the numerical aperture is improved while the sample is not damaged.
On the basis of this structure, the different indexes described in claim 2 can be realized by changing the glass material, changing the positions and parameters of the lenses and the lens group, and changing the observation medium (such as water, glycerin, cedar oil, etc.), and since the example 1 given above already belongs to the stricter index in the range, it is no problem to adjust it to the remaining value in the range.
The lens data for example 1 are shown in the following table:
table 1 example 1 lens data
The two-photon light field calculation microscope objective adopts a form of split aperture design, and has the following advantages: 1. the optical design pressure is greatly reduced. After the large numerical aperture is divided into the sub-apertures with small numerical apertures, the difficulty of optimizing the optical aberration of each sub-aperture is greatly reduced. 2. The robustness to scattering is improved. When the large numerical aperture is used for illumination excitation, because of the illumination with a large angle, scattering is easy to generate, a deeper sample cannot be penetrated, and the imaging quality is reduced; and the small numerical aperture can reduce the light incidence angle, is more robust to scattering, not only can improve the penetration depth, but also can ensure the imaging quality. 3. And the signal-to-noise ratio of the system is improved. In the three-dimensional body excitation imaging process, the light spot with a larger volume can be formed by the small numerical aperture, so that more photon numbers are collected, the signal-to-noise ratio of the system is improved, and the signal-to-noise ratio can be further improved for the multiplexing of axial information.
In addition, each lens in the objective lens is spherical, and is coaxially arranged, so that the processing, the assembly and the adjustment are convenient; the lens material is common commercial glass, so that the purchasing difficulty and the manufacturing cost of the optical system material are reduced.
In consideration of energy and stability, a lens material with high transmittance is selected in the design process, so that the thickness of the lens is reduced while high imaging quality is ensured, and the overall energy loss of the system is ensured to be low; the diameter of the gluing surface of the designed double-gluing lens is less than 75mm in consideration of the stability of the gluing process.
The conventional 'excitation-collection' common objective mechanism is mostly used in the existing microscope objective, so that the small numerical aperture is needed for improving the penetration depth, the large numerical aperture is needed for improving the imaging resolution, and the irreconcilable contradiction exists. The design innovatively adopts an excitation and collection asymmetric objective structure, ensures penetration depth by regional scanning with small numerical aperture, collects energy with large numerical aperture and improves resolution ratio, thereby solving the contradiction. In addition, due to the reverse design, the objective lens is actually an image space telecentric system, so that the image plane illumination is very uniform.
The imaging quality analysis of the two-photon light field calculation microscope objective formed in example 1 is shown in fig. 2 to 4. As shown in fig. 2, the modulation transfer function MTF value of the two-photon light field calculation microscope objective is higher in the full field range of the working band, and a larger margin is reserved for subsequent installation and adjustment; as shown in fig. 3, the diffuse spot root mean square radius of the two-photon light field calculation microscope objective is less than 10 μm in the working waveband full-view field range; as shown in FIG. 4, the field curvature of the two-photon light field calculation microscope objective lens is less than + -12 um in the working band full view field range, and the relative distortion is less than + -0.3% in the working band full view field range.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (8)
1. A two-photon light field calculation microscope objective is characterized in that the two-photon light field calculation microscope objective is of an asymmetrical structure of an excitation aperture and a collection aperture, adopts a split aperture design, excites fluorescence with a small numerical aperture, and collects energy with a large numerical aperture; the two-photon light field calculation microscope objective comprises a beam expander group BE, a dichroic mirror D, a water mirror O and a medium W which are sequentially arranged along an optical axis; the beam expander set BE comprises a focusing set G1 and a collimating set G2 and is used for focusing incident parallel light once and then expanding and collimating the incident parallel light, the dichroic mirror D is used for dividing exciting light and excited fluorescent light into respective light paths, the water mirror O comprises a similar beam expander set G3 and a focusing set G4 and is used for expanding the incident light and then focusing and imaging the expanded incident light, and the medium W is used for increasing the numerical aperture;
the focusing lens group G1 comprises a first convex-concave lens L1, a first biconvex lens L2, a second biconvex lens L3 and a second convex-concave lens L4 which are sequentially arranged along the optical axis direction.
2. The two-photon light field calculation microobjective of claim 1, wherein the numerical aperture of the two-photon light field calculation microobjective is 0.3-1.0, the object field diameter is 1mm-10mm, the object lateral resolution is 0.3 μm-2 μm, the excitation light wavelength range is 400nm-1000nm, the fluorescence wavelength range is 460nm-700nm, and the axial resolution is 1 μm-10 μm.
3. A two-photon light field computing microscope objective lens according to claim 1, characterized in that the collimator group G2 comprises a first double concave lens L5, a first concave-convex lens L6 and a third double convex lens L7 arranged in the optical axis direction in this order.
4. The two-photon light field computing microobjective of claim 1, wherein the beam expander group G3 comprises a fourth biconvex lens L8, a second biconcave lens L9, a third biconcave lens L10, a second meniscus lens L11 and a fifth biconvex lens L12 arranged in the optical axis direction in sequence.
5. A two-photon light field computing microobjective as claimed in claim 1, wherein the focusing lens group G4 comprises a first convex flat lens L13, a first flat concave lens L14, a sixth biconvex lens L15 and a second convex flat lens L16 arranged in this order along the optical axis direction.
6. A two-photon light field computing microscope objective lens according to claim 1, wherein the beam expander group BE is capable of effectively adjusting the beam diameter and the divergence angle such that the beam exiting from the beam expander group BE has a smaller divergence angle as better as possible under the condition of satisfying the size matching the machinable dichroic mirror D.
7. A two-photon light field computing microscope objective lens according to claim 1, characterized in that the beam expander group BE is capable of correcting aberrations mainly including spherical aberration and coma in combination with the water mirror O, so that the resolution of each field approaches the diffraction limit.
8. A two-photon light field computing microscope objective lens as claimed in claim 1, wherein the medium W is water, cedar oil or cedar oil.
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| CN111258044B (en) * | 2020-02-18 | 2021-05-04 | 清华大学 | Mirror scanning light field-based 4 pi-like microscopic imaging method |
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| JP2010145950A (en) * | 2008-12-22 | 2010-07-01 | Nikon Corp | Liquid-immersion objective lens and microscope including the same |
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| CN109633883A (en) * | 2019-01-23 | 2019-04-16 | 浙江大学 | A kind of high-resolution fluorescence microscopy endoscope objective lens of big visual field |
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