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大视场双光子显微成像系统的设计需要在光学分辨率和成像视场之间进行权衡,最终以具体成像需求为判断依据取得两者间的平衡。实现大视场、高分辨的双光子成像的最大难度便在于如何在保证分辨率的情况下增加成像视场。
对于双光子成像系统,成像物镜的数值孔径(Numerical Aperture, NA)决定了该系统成像的衍射极限分辨率 [1],即
$$ \omega_{xy}=\left\{\begin{array}{l} \dfrac{0320 \lambda}{\sqrt{2} NA}, NA \leqslant 0.7 \\ \dfrac{0.325 \lambda}{\sqrt{2} NA^{0.91 }}, N A>0.7 \end{array}\right. $$ (1) $$ \omega_{z}=\frac{0.552\lambda}{\sqrt{2}}\left[\frac{1}{n-\sqrt{n^{2}-NA^{2}}}\right] $$ (2) 式中:ωxy和ωz分别为横向和轴向的分辨率;λ为入射光波长;NA为数值孔径;n为物镜下介质折射率。要实现近衍射极限分辨率的大视场成像,首先需要大孔径入射光束使有效NA达到物镜标定NA,获得衍射极限分辨率。物镜有效数值孔径可表示为 [30]:
$$ NA_{e ff}=\frac{d_{e ff}}{d_{p upil}} N A $$ (3) 式中:NAeff为有效数值孔径;deff 为实际后背孔径出入射光斑直径;dpupil为物镜后背孔径处入瞳直径;NA为物镜标准数值孔径大小。只有当NAeff = NA,即deff = dpupil时,在不考虑像差等影响的理想情况下,物镜才能实现近衍射极限的高分辨成像。
物镜放大倍数和视场数将决定成像系统的最大成像视场(Field-of-view, FOV),其视场直径为 [30-32]:
$$ F O V=\frac{F N}{M}=2 \times F L \times \tan \theta $$ (4) 式中:FN为物镜的视场数;M为物镜的放大倍数;FL为物镜的前焦距;θ为光束进入物镜后背孔径的角度。该公式前部分
$ {FOV}=\dfrac{FN}{M} $ 说明,物镜的成像视场由FN视场数和M放大倍数决定,即物镜的场数FN越大,放大倍数越小,其支持的最大成像视场越大。该公式后部分$ F O V=2 \times F L \times \tan \theta$ 说明,当物镜的焦距越长,后备孔径处入射光束角度越大,其支持的成像视场越大。在双光子系统中,可用一个光学量来同时衡量各光学元件(系统)的衍射极限分辨率和视场大小,人们称之为光学不变量I [30],其表达式为:
$$ I=NA \frac{FN}{2 M} $$ (5) 式中:数值孔径NA决定了该光学元件(系统)的衍射极限分辨率;视场数FN和放大倍数M决定了该光学元件(系统)的成像视场。对于传统的双光子成像显微镜,一般都是在牺牲成像视场的情况下增大数值孔径NA,即在直径为500 µm左右的视场内保证几百纳米的高分辨成像。而对于大视场双光子成像系统,不仅要保证数值孔径NA接近传统双光子成像系统,而且需要保证成像视场相对提升数倍。所以大视场高分辨的双光子成像需要对成像系统中各元件进行大光学不变量的挑选或特殊设计。
2018年,Jonathan R. Bumstead等提出了利用光学不变量的原理来设计大视场双光子成像系统 [30]。
在理想双光子成像系统中,可分别计算出系统中各光学元件的光学不变量。对各光学元件的光学不变量的大小对比后,选择该光学成像系统中光学不变量最小的一个或几个光学元件进行替换升级。一般来说,由于成像物镜的设计难度和设计成本较高,所以双光子成像系统中的最大光学不变量是由商业可选的高性能大视场成像物镜决定。在选定物镜的情况下,通过替换或设计其他各光学元件以充分利用物镜的成像性能,从而实现更大视场的高分辨率双光子成像。
具体来说,在设计大视场双光子成像系统时,可将其大致分为三部分进行分块设计优化,分别为成像物镜、扫描中继系统与荧光收集系统。
大视场成像物镜一般选择10、4倍等放大倍数较小的型号。选定几款低放大倍数的双光子成像物镜,对比这些物镜的光学不变量之后选择最大光学不变量的一款,以保证在增大视场的情况下尽可能提高最终成像分辨率。如图1(a)所示,物镜入射光学不变量为入射光束半径和入射角度正弦值之积r0sinθ0,出射光学不变量为聚焦半光锥角的正弦值和成像视场半径的乘积F0sinα0。该文认为对于整个成像系统,其光学不变量最大的限制来自于物镜,物镜限制了除物镜外双光子成像系统所能达到的最大光学不变量,而物镜的出射光学不变量决定了最后整个成像系统能达到的最大成像视场。
图 1 光学不变量的定义。(a) 物镜的光学不变量;(b) 扫描中继的光学不变量; (c) 荧光收集系统的光学不变量
Figure 1. Definition of optical invariants. (a) Optical invariants of the imaging objectives; (b) Optical invariants of the scanning relay; (c) Optical invariants of the fluorescence collection system
设计扫描中继时,他们对现有的光学透镜进行光学不变量计算后,在不影响系统结构的情况下选择了光学不变量等于或超过物镜的透镜元件。扫描中继可视作一个整体进行光学不变量分析,如图1(b)所示,其入射光学不变量等于出射光学不变量,即:
$$ r \sin \theta=r^{\prime} \sin \theta^{\prime} $$ (6) 式中:r和r'分别为入射光束和出射光束的半径;θ和θ'分别为入射光束和出射光束的最大扫描角度。通常双光子系统中含两个扫描中继,从X扫描振镜共轭到Y扫描振镜的第一个扫描中继,以及Y振镜共轭到物镜后背孔径处的由扫描镜和与物镜配套的管镜组成的第二个扫描中继。
荧光收集系统的光学不变量同样应等于或大于选定物镜的光学不变量。荧光收集系统中光学元件包括二向色镜(Dichroic mirror, DM)、收集透镜(系统)(Collective lens, CL),荧光收集系统的光学不变量主要由收集透镜(系统)决定,因此可视为单透镜分析。如图1(c)所示,入射光学不变量r’sinθ’等于出射光学不变量FFluosinαFluo,r’、θ、FFluo、αFluo分别为入射光束半径、入射角、荧光收集设备靶面半径和出射半光锥角。
Bumstead等最终实现视场直径为7 mm,横向分辨率<1.7 μm,轴向分辨率<28 μm的高分辨大视场双光子成像。其成像系统和血管成像结果如图2所示。
Advances of large field-of-view two-photon microscopy system (invited)
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摘要: 双光子显微成像具备高分辨率、天然层析能力和大穿透深度等特点,在活体动物成像中发挥着重要作用。然而,如何在维持高分辨率的条件下,扩大双光子的成像视场,来满足生物医学中对大规模动态反应的监测需求,一直以来都是光学显微成像领域的难点,也是科研关注的重点。综述了大视场双光子成像技术的研究进展。首先介绍了双光子显微成像系统的产生背景和设计原理,并从光学不变量的角度阐述了实现大视场双光子成像的理论基础。然后重点回顾了现有的几种大视场双光子成像方法,分别包括了扫描中继系统的边缘像差校准、高通量物镜的设计研发和自适应光学方法的使用。基于双光子成像的高时间和空间分辨特性,大视场双光子成像技术将成为一种在脑科学等需介观高分辨成像领域的应用中实现大区域动态监测的强有力的工具。Abstract: Two-photon microscopy (TPM) imaging has the characteristics of high resolution, natural chromatography capability and large penetration depth, and plays an important role in the imaging of living animals. How to enlarge the field-of-view (FOV) of TPM while maintaining the high resolution to monitor large-scale dynamic responses in biomedical applications especially brain science, however, remains challenging. In this paper, the recent progress of large-FOV two-photon imaging technology is reviewed. The theoretical basis of achieving large-FOV TPM is elaborated from the perspective of optical invariant. Large- FOV TPM methods can be divided into three categories: FOV-edge aberration calibration with scanning relay engines, the design and manufacture of high-throughput objectives and correcting aberrations with adaptive optics. These methods have highly strengthened the capability of TPM used in large scale biomedical imaging. If further improved especially the imaging speed, large-FOV TPM will have great potential to contribute the development of life science and broaden the cognitive of large-scale biological activities. Large-FOV TPM, based on its outstanding spatial and temporal resolution, will become a powerful tool for dynamic monitoring across large-area in some applications that requires high resolution and mesoscale imaging simultaneously.
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Key words:
- large field-of-view /
- two-photon microscopy /
- imaging objective lens /
- aberration /
- adaptive optics /
- optical invariant
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图 3 透镜组优化大视场扫描离轴像差实现大视场双光子成像系统。(a) 成像系统图;(b) 视场边缘位置和中心位置的点扩散函数,由0.5 µm荧光小球测得;(c) 开双颅的清醒、头部固定的小鼠左右脑半球的大视场双光子小动脉血管成像(波长800 nm,最大强度投影)
Figure 3. Large field-of-view two-photon microscopy by optimizing the off-axis aberrations using lens series. (a) Optical layout; (b) Experimental measurements of the PSF (point-spread-function) as a function of FOV, measured by 0.5 µm fluorescence beads; (c) Large-FOV two-photon image (λ= 800 nm, max intensity project) of vasomotion in cortical arterioles across both hemispheres of an awake, head-fixed mouse through dual transcranial windows
图 4 大视场双光子随机扫描显微系统。(a) 成像系统图;(b) 视场边缘位置和中心位置的点扩散函数,由0.5 µm荧光小球测得;(c) 大视场成像麻醉thy-1小鼠荧光蛋白(最大强度投影)和虚线区域的细节图
Figure 4. Large-FOV two-photon random access microscopy. (a) Optical layout; (b) PSF at middle position and edge position of the FOV, measured by 0.5 µm microbeads; (c) Large-FOV two-photon image of fluorescent proteins in anesthetized thy-1 mice (max intensity project) and higher magnification image (dashed box in the large-FOV image)
图 5 大视场多区域双光子扫描显微镜。(a) 成像系统图;(b) 视场边缘位置和中心位置的点扩散函数,由0.5 µm荧光小球测得;(c) 大视场成像检测转基因小鼠的神经元活动,该小鼠表达GCaMP6 s钙离子荧光指示剂。(d) 分割上图产生的5, 361 个活动神经元;(e) 同时的双区域神经成像
Figure 5. Large field-of-view and multi-region two-photon microscopy. (a) Optical layout; (b) PSF at middle position and edge position of the FOV, measured by 0.2 µm microbeads; (c) Large-FOV imaging was used to examine neuronal activity of a transgenic mouse expressing the genetically encoded fluorescent calcium indicator GCaMP6 s; (d) Segmenting the image sequence can yield 5,361 active neurons; (e) Simultaneous two region imaging to monitor neuronal activity between two cortical visual areas
图 6 使用自适应光学提高双光子成像视场。(a) 原理图;(b) 成像光路图;(c) thy-1小鼠脑片的大视场成像图(最大强度投影);(d) 图 (c)左下角标示位置荧光信号通过自适应光学技术的提升
Figure 6. Extending the field of view of two-photon microscopy using adaptive optics. (a) Schematic diagram; (b) Imaging optical path diagram; (c) Large-FOV two-photon image of the brain slice of thy-1 mice (max intensity project); (d) Comparison before and after adaptive optics correction in the yellow solid area of Fig. (c)
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