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NIR-II共聚焦显微系统的激发光与发射光是逐渐由可见光向NIR-I再向NIR-II扩展的,随着光波长的增加,激发光的散射减小,能更好地聚焦在目标点上;当发射光在NIR-IIx、NIR-IIa或NIR-IIb波段时,信号收集过程中能收集到更高比例的弹道光子,从而提升成像质量和成像深度。第二部分中所展示的工作都是基于荧光发射的NIR-II共聚焦显微系统,此类共聚焦显微系统依赖于NIR-II荧光发射的探针,成像深度很大程度上受限于探针的亮度和吸收发射波段。反射式共聚焦显微镜采用单个波长激发,收集同一个波长反射回来的信号,可在一定程度上摆脱对探针的依赖,激发光聚焦样品之后收集反射的信号,在延长信号光波长的同时相应延长激发光波长,使其在生物组织大深度成像中具有天然优势。
2018年,Chris Xu组[43]将NIR-II激光引入反射式共聚焦系统(RCM)。RCM面临的一个挑战是光束路径中光学元件的杂散反射,他们在光路中引入偏振来滤除非样品上的反射信号。如图8(a)所示,他们使用5 mm2的方形λ/4波片代替普通的盖玻璃作为颅窗的盖玻片,光路中的杂散反射光返回经过λ/2波片后仍然为P偏振光;而只有两次经过λ/4波片的样品上的反射信号再经过λ/2波片之后转化为S偏振光后才能在PBS中反射并被PMT1或PD探测收集。他们使用1650 nm的脉冲激光器作为入射光源,并使用InGaAs PMT(H10330 C-75,滨松)作为探测器。系统中,后向散射光通过一个75 mm的透镜重新聚焦到作为共聚焦针孔的25 µm直径的多模光纤中,在脑表面以下1.2~1.3 mm的深度观察到了海马体的神经纤维(图8(c))。在这项工作中,Chris Xu组还使用反射式共聚焦显微系统展示了在1310 nm、1610 nm和1630 nm的连续波和1650 nm的脉冲激光照射下小鼠的白质共聚焦图像,并利用不同波长采集图像,证实了长波长照射下反射式共聚焦显微成像效果更好(图8(b))。
图 8 (a) NIR-II反射式共聚焦显微镜示意图;(b) 相同功率,不同波长连续脉冲激光(1 310 nm, 1 610 nm, 1 630 nm和1 650 nm)激发下白质的RCM共聚焦图像;(c) 不同深度下的活体RCM图像[43](比例尺:30 µm)
Figure 8. (a) Schematic illustration of the NIR-II reflectance confocal microscope; (b) Confocal images of white matter under CW and pulsed laser illumination with the same power at 1310 nm, 1610 nm, 1630 nm, and 1650 nm; (c) In vivo reflectance confocal microscope images at various depths[43] (Scale bars: 30 μm)
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SNSPD是一类用单个光子打破库珀对从而产生电流电压信号的量子极限超导光检测器[43]。Kadin等人于1996年观察到了超导薄膜中的非平衡热点效应,并以此为基础首次提出了SNSPD的概念[44]。2001年,俄罗斯Goltsman团队利用NbN薄膜制备的超导单光子纳米线实现了810 nm波长光的探测,成功证明了SNSPD的概念[45]。自此,SNSPD在各种应用方面的巨大潜力吸引了世界各地的研究人员。研究人员们通过改进材料、设计、制造工艺和理论等,使SNSPD的性能得到了有效的提高。其中,系统探测效率(SDE)是最受关注的性能,它通常可以被表示为光耦合效率、吸收效率和本征探测效率三者的乘积。成熟的光学技术(光学薄膜、透镜光学、光学仿真计算等)和超导薄膜及微纳加工技术,使得以上三个因素都能够做到近乎理想[46]。在2019年的罗彻斯特相干和量子光学会议上,Reddy等人报道了SDE为98%的SNSPD,这是SNSPD当时所达到的最高效率[47]。此后,上海微系统所、美国NIST、荷兰TU Delft三个团队都先后报道了在1550 nm波段SDE接近100%的SNSPD[48-50]。与此同时,SNSPD对更长波长光信号(1 900~2 000 nm)也有较高灵敏度(检测效率>50%),这种长波长高探测效率器件的出现使得近红外二区的生物成像应用得到了拓展。除了探测效率的明显优势,SNSPD的快速时间响应(~109 ps)、低定时抖动(~50 ps)和低暗计数率(<100 cps)等特点都使其十分有利于NIR-II生物成像。
2020年,中国科学院深圳先进技术研究院郑炜研究员团队[51]利用了SNSPD高灵敏度的特点开发了一种NIR-II荧光共聚焦介观成像系统。如图9(a)所示,波长为808 nm的飞秒激发光被扩束后由二色镜反射到二维振镜扫描仪上进行扫描,相比于共聚焦显微系统,此系统将原本用于聚焦的短焦距显微物镜换成了单一的扫描透镜(LSM02-BB,f=18 mm,Thorlabs),从而创造了一个更大的平面成像场。相比于显微成像,介观成像中聚焦光斑大,功率密度低,激发出的荧光信号更加微弱,需要用高探测效率的探测器即SNSPD弥补。文中所使用的SNSPD在2.5 K温度下工作,对1064 nm (无偏振)波长的检测效率大于50%,暗计数率仅100次/s,它为实现同时具备较大视场、高分辨率、大穿透深度及光学切片能力的介观成像提供了探测的基础。在这项工作中,他们开发出了FOV为7.5 mm×7.5 mm、横向分辨率为6.3 µm、成像深度可达小鼠颅骨表面以下2.5 mm的介观成像系统,并利用该系统对小鼠进行了活体三维脑血管成像,实现了局部缺血的动态脑血管成像(图9(b))。
2021年,Chris Xu教授团队[52]基于与图4、5类似的荧光共聚焦系统光路将长波长发射的荧光探针PbS/CdS量子点(激发波长为1310 nm,荧光发射波长>1600 nm)与定制的SNSPD(检测范围在1300~2 000 nm)相结合,使用低功率连续波激光进行激发,在开颅成年小鼠体内获得了约1.7 mm深度的脑血管共聚焦荧光显微图像(图10)。在这项工作中,量子点荧光探针的激发和发射波长都很长,NIR-II激发、发射的共聚焦荧光显微成像得以首次实现,并达到了可以与双光子成像相媲美的成像深度。同时,他们使用了在长波长处有着更高灵敏度的探测器SNSPD,达到了远超以往的NIR-II荧光共聚焦成像深度。
2022年,戴宏杰院士课题组[53]合成了发射峰在1880 nm的生物相容核壳PbS/CdS量子点,系统中1700 nm以上波长的荧光被耦合进单模光纤并采用高灵敏度的SNSPD探测,其余光路结构与图4、5所示的荧光共聚焦光路基本一致。与较短波长的激光(800 nm、1000 nm、1319 nm和1540 nm)相比,文中所使用的1650 nm的长波长激发光在生物组织中的散射进一步降低,有助于在深层组织中实现良好聚焦;NIR-IIc区发射光在生物组织中的散射进一步降低,可以实现大深度穿颅成像。最终,他们使用迄今为止最长激发和发射波长的共聚焦荧光显微术,实现了穿颅条件下小鼠脑血管约1100 µm的成像深度(图11(a)~(b)),并在小鼠体内腹股沟淋巴结中实现了直径约6.6 µm的高内皮小静脉、以及淋巴结中CD169+巨噬细胞和CD3+T细胞的高分辨率成像,极大推动了近红外共聚焦显微术的发展。如图11(c)所示,这项工作中还对比了在NIR-IIb或NIR-IIc窗口内使用PMT或SNSPD作为探测器时,小鼠脑部不同深度血管的荧光共聚焦显微图像,进一步验证了长波长激发光以及高灵敏度SNSPD在共聚焦显微成像中的优势。至此,NIR-II荧光共聚焦显微成像系统得到了进一步的发展。文中所介绍的所有NIR-II荧光共聚焦显微成像系统的具体参数如表1所示。
图 11 NIR-IIc窗口无创活体共聚焦显微镜穿颅观察小鼠脑血管[53]。(a) NIR-IIc窗口小鼠穿颅共聚焦显微成像示意图;(b) 小鼠脑部血管的穿皮三维共聚焦图像;(c) 在NIR-IIb或NIR-IIc窗口内使用PMT或SNSPD作为探测器时,小鼠脑部不同深度血管的高分辨率荧光共聚焦显微图像
Figure 11. Non-invasive in vivo confocal microscopic imaging of intact mouse head in NIR-IIc window[53]. (a) Schematic of intact mouse confocal microscopic head imaging in NIR-IIc window; (b) Three-dimensional volumetric images of blood vessels in an intact mouse head visualized through the scalp; (c) High-resolution fluorescence confocal images of blood vessels at various depths through intact mouse head imaged with a PMT or SNSPD in NIR-IIb or NIR-IIc window
表 1 所引用文献中NIR-II共聚焦显微成像系统的相关参数
Table 1. Related parameters of the NIR-II confocal microscopic imaging system introduced in this paper
Applications Fluorescence probe Excitation (wavelength/power) Scanning mode Scanning rate Detector Emission Imaging depth Imaging of brain
tissues sections[8]IR-FGP 785 nm (~160 mW) Stage scanning 2.5 ms/pixel H12397-75 PMT 1 050-1 300 nm 170 µm Imaging of cerebral vessels in mouse ex vivo[33] p-FE 785 nm (~30 mW) Stage scanning 7.5 min/frame H12397-75 PMT 1 100 LP 1 350 µm Galvanometer scanning 2 s/frame Imaging of tumor in vivo[33] p-FE 785 nm (~30 mW) Stage scanning 15 min/frame H12397-75 PMT 1 100-1 300 nm 220 µm CNTs 1 500-1 700 nm Imaging of cerebral vessels in mouse in vivo (craniotomy)[34-35] ICG 793 nm (~40 mW) Galvanometer scanning 20 µs/pixel H12397-75 PMT 1 000 LP 300 µm TB1 793 nm (~70 mW) 10 µs/pixel 1 000 LP 800 µm Imaging of cerebral vessels in non-human primates in vivo
[41]ICG 793 nm (~20 mW) Galvanometer scanning 20 µs/pixel H12397-75 PMT 900 LP 470 µm Imaging of tumor in vivo[42] PEG-CSQDs 785 nm (~40 mW) Stage scanning 5-7 min/frame H12397-75 PMT 1 500 LP 1.2 mm Imaging of cerebral vessels in mouse in vivo (craniotomy)[52] PbS/CdS QDs 1 310 nm (≤25 mW) Galvanometer scanning 1-50 s/
frameSNSPD 1 300-1 700 nm 1.7 mm Imaging of cerebral vessels in mouse in vivo (through the scalp)[53] P3-QDc 1 650 nm (~28.5 mW) Galvanometer scanning 5-20 s/frame SNSPD 1 800-2 000 nm 1.1 mm
Progress and application of near-infrared II confocal microscopy (invited)
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摘要: 共聚焦显微镜具有较高的空间分辨率和信号背景比,能对生物样品进行三维层析成像,在医学与生物学领域有着广泛的应用。近红外二区(NIR-II,900~1 880 nm)波段的光在生物组织中具有适中的吸收、较低的散射,以及非常弱的生物组织自发荧光,因此,NIR-II荧光活体成像具有大深度、高对比度等优势。点激发、点探测的NIR-II共聚焦显微技术结合了上述二者的优势,在大深度生物成像中具有高空间分辨率和高信号背景比等优点,因此在生物医学领域得到了广泛应用。此综述将从NIR-II共聚焦显微技术的原理出发,阐述其发展进程、以及基于此项技术开展的生物医学成像应用,探讨NIR-II共聚焦显微技术未来的改进和发展方向。Abstract: Confocal microscopy has high spatial resolution and signal to background ratio, possessing the capability of three-dimensional tomography of biological samples, and thus has been widely used in medicine and biology areas. Light in near-infrared II (NIR-II, 900-1 880 nm) regions fulfils moderate absorption, low scattering in biological tissues, and weak autofluorescence of biological tissues. Therefore, NIR-II in vivo fluorescence imaging has the advantages of large depth and high contrast. Point-excitation and point-detection based NIR-II confocal microscopy combines the advantages of the two technologies mentioned above and features high spatial resolution and high signal to background ratio in large-depth biological imaging. Therefore, it has been widely used in the biomedical fields. This review summarizes the principle and the development progress of NIR-II confocal microscopy and the application of biological imaging based on it. The future improvement and development directions of NIR-II confocal microscopy are also discussed.
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Key words:
- confocal microscopy /
- near-infrared II /
- in vivo bioimaging
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图 1 IR820标记的小鼠大脑血管的NIR-I荧光共聚焦显微成像[26]。(a) 不同垂直深度下(0~500 µm)的小鼠脑血管荧光显微镜图像;(b) 小鼠脑血管的三维重建图像,比例尺:50 µm
Figure 1. NIR-I fluorescence confocal microscopic images of blood vessels of the mouse brain stained with IR820[26]. (a) Fluorescence microscopic images of blood vessels in the mouse brain at various vertical depths (0-500 µm); (b) 3D reconstructive images of blood vessels of the mouse brain. Scale bar: 50 μm
图 2 NIR-II转盘式共聚焦显微技术的应用[31]。(a) NIR-II转盘式共聚焦显微技术的光路示意图;(b) 宽场(左)和转盘式(右)共聚焦显微镜对(186±48) nm NIR荧光珠成像的比较,比例尺:1 µm;(c) 荧光珠的宽场(上) 和共聚焦(下)显微成像的层析图,比例尺:2 µm
Figure 2. The application of NIR-II spinning-disc confocal microscopy[31]. (a) Optical layout of the NIR-II spinning-disc confocal microscopy; (b) Comparison of wide-field (left) and spinning-disc(right) confocal microscope imaging for (186±48) nm NIR fluorescent beads. Scale bar: 1 µm; (c) Z-stack projection of wide-field (top) and confocal (bottom) microscopic images of the fluorescent beads. Scale bar: 2 µm
图 3 (a) 移动样品台扫描的NIR-II荧光共聚焦显微装置的光路图[32]; (b) 小鼠脑血管的离体NIR-II荧光共聚焦大面积(3000 µm×2 000 µm)成像[33]; (c) 小鼠脑血管的离体NIR-II荧光共聚焦小体积(左侧,200 µm×200 µm×200 µm)和 (d) 大体积(右侧,400 µm×400 µm×400 µm) 3D重构图像[33];(e) NIR-II荧光共聚焦显微镜下肿瘤组织和血管的双色成像[33]
Figure 3. (a) Optical diagram of the stage-scanning NIR-II fluorescence confocal microscopic device[32]; (b) Ex vivo NIR-II fluorescence confocal imaging in large area (3000 µm×2 000 µm) of brain in a mouse injected with p-FE[33]; (c) 3D reconstruction of ex vivo NIR-II fluorescence confocal imaging of vasculatures in brain of mouse within a small volume (left side, 200 µm×200 µm×200 µm) and (d) a large volume (right side, 400 µm×400 µm×400 µm)[33]; (c) Two-color fluorescence confocal microscope imaging of a tumor tissue and blood vessels in the NIR-II window[33]
图 4 (a) NIR-II荧光共焦扫描显微成像系统的原理图;(b) 活体NIR-II荧光共聚焦扫描显微镜下小鼠在40~300 µm不同深度的开颅脑血管成像[34]
Figure 4. (a) Schematic illustration of NIR-II fluorescence confocal scanning microscopic imaging system; (b) In vivo NIR-II fluorescence confocal scanning microscopic imaging of cerebrovasculature in a mouse with craniotomy at various depths from 40 to 300 µm[34]
图 6 (a) NIR-II荧光共聚焦显微成像系统示意图(红色箭头:微调。蓝色箭头:旋转。绿色箭头:粗调整。右侧图是激发光路和成像光路);(b) NIR-II荧光共聚焦显微镜下恒河猴大脑血管的大深度活体成像[41](比例尺:100 µm)
Figure 6. (a) The schematic illustration of the NIR-II fluorescence confocal microscopic imaging system. Red arrows: fine adjustment. Blue arrows: rotation. Green arrows: coarse adjustment. The excitation and imaging light paths are showed on the right panel; (b) In vivo NIR-II fluorescence confocal microscopic imaging of cerebral blood vessels of the rhesus macaque with large penetration depth[41](Scale bar: 100 µm)
图 7 (a) NIR-IIb窗口小鼠肿瘤活体无创荧光共聚焦显微成像[42](比例尺:500 µm);(b)~(c)180µm深度下肿瘤血管的高分辨率荧光共聚焦图像:(b) 比例尺:200 µm;(c) 比例尺:50 µm
Figure 7. (a) In vivo noninvasive NIR-IIb fluorescence confocal imaging of the mouse tumor[42]. Scale bar: 500 µm. (b)-(c) High-resolution fluorescence confocal imaging of tumor vessels at a depth of 180 μm: (b) Scale bar: 200 µm; (c) Scale bar: 50 µm
图 8 (a) NIR-II反射式共聚焦显微镜示意图;(b) 相同功率,不同波长连续脉冲激光(1 310 nm, 1 610 nm, 1 630 nm和1 650 nm)激发下白质的RCM共聚焦图像;(c) 不同深度下的活体RCM图像[43](比例尺:30 µm)
Figure 8. (a) Schematic illustration of the NIR-II reflectance confocal microscope; (b) Confocal images of white matter under CW and pulsed laser illumination with the same power at 1310 nm, 1610 nm, 1630 nm, and 1650 nm; (c) In vivo reflectance confocal microscope images at various depths[43] (Scale bars: 30 μm)
图 11 NIR-IIc窗口无创活体共聚焦显微镜穿颅观察小鼠脑血管[53]。(a) NIR-IIc窗口小鼠穿颅共聚焦显微成像示意图;(b) 小鼠脑部血管的穿皮三维共聚焦图像;(c) 在NIR-IIb或NIR-IIc窗口内使用PMT或SNSPD作为探测器时,小鼠脑部不同深度血管的高分辨率荧光共聚焦显微图像
Figure 11. Non-invasive in vivo confocal microscopic imaging of intact mouse head in NIR-IIc window[53]. (a) Schematic of intact mouse confocal microscopic head imaging in NIR-IIc window; (b) Three-dimensional volumetric images of blood vessels in an intact mouse head visualized through the scalp; (c) High-resolution fluorescence confocal images of blood vessels at various depths through intact mouse head imaged with a PMT or SNSPD in NIR-IIb or NIR-IIc window
图 12 (a) NIR-II共聚焦荧光寿命显微成像系统的光路图;(b) 静脉注射TB1后的小鼠大脑血管的活体NIR-II FLIM图像;(c) 在 (b) 箭头处测量的荧光衰减曲线,得到TB1在血管中的荧光寿命为1.5 ns[35]
Figure 12. (a) A simplified optical diagram of NIR-II confocal fluorescence lifetime microscopic imaging system; (b) An in vivo NIR-II FLIM image of cerebral vessels in mouse, which was intravenously injected with TB1 dots; (c) Fluorescence decay curves measured at the arrow in (b), showing the fluorescence lifetime of TB1 dots in vessels is 1.5 ns[35]
表 1 所引用文献中NIR-II共聚焦显微成像系统的相关参数
Table 1. Related parameters of the NIR-II confocal microscopic imaging system introduced in this paper
Applications Fluorescence probe Excitation (wavelength/power) Scanning mode Scanning rate Detector Emission Imaging depth Imaging of brain
tissues sections[8]IR-FGP 785 nm (~160 mW) Stage scanning 2.5 ms/pixel H12397-75 PMT 1 050-1 300 nm 170 µm Imaging of cerebral vessels in mouse ex vivo[33] p-FE 785 nm (~30 mW) Stage scanning 7.5 min/frame H12397-75 PMT 1 100 LP 1 350 µm Galvanometer scanning 2 s/frame Imaging of tumor in vivo[33] p-FE 785 nm (~30 mW) Stage scanning 15 min/frame H12397-75 PMT 1 100-1 300 nm 220 µm CNTs 1 500-1 700 nm Imaging of cerebral vessels in mouse in vivo (craniotomy)[34-35] ICG 793 nm (~40 mW) Galvanometer scanning 20 µs/pixel H12397-75 PMT 1 000 LP 300 µm TB1 793 nm (~70 mW) 10 µs/pixel 1 000 LP 800 µm Imaging of cerebral vessels in non-human primates in vivo
[41]ICG 793 nm (~20 mW) Galvanometer scanning 20 µs/pixel H12397-75 PMT 900 LP 470 µm Imaging of tumor in vivo[42] PEG-CSQDs 785 nm (~40 mW) Stage scanning 5-7 min/frame H12397-75 PMT 1 500 LP 1.2 mm Imaging of cerebral vessels in mouse in vivo (craniotomy)[52] PbS/CdS QDs 1 310 nm (≤25 mW) Galvanometer scanning 1-50 s/
frameSNSPD 1 300-1 700 nm 1.7 mm Imaging of cerebral vessels in mouse in vivo (through the scalp)[53] P3-QDc 1 650 nm (~28.5 mW) Galvanometer scanning 5-20 s/frame SNSPD 1 800-2 000 nm 1.1 mm -
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