Advances in high spatiotemporal resolution fluorescence microscopic imaging technique based on point scanning

Pan Bin-Xiong; Gong Cheng; Zhang Peng; Liu Zi-Ye; Pi Peng-Jian; Chen Wang; Huang Wen-Qiang; Wang Bao-Ju; Zhan Qiu-Qiang

doi:10.7498/aps.72.20230912

Abstract

Laser point-scanning fluorescence microscopy serves as an indispensable tool in the life science research, owing to its merits of excellent resolution, high sensitivity, remarkable specificity, three-dimensional optical-sectioning capability, and dynamic imaging. However, conventional laser point-scanning fluorescence microscopy confronts a series of challenges in the rapidly evolving field of life sciences, because of the limitations imposed by optical diffraction and point scanning detection. Over the past two decades, substantial advancements have been made in super-resolution fluorescence microscopic imaging techniques. Researchers have developed various high spatial and temporal resolution point-scanning microtechniques, which hold great significance for biological optical imaging and other relevant applications. Regrettably, there are still few review articles covering the recent progress of this field. It is essential to provide a comprehensive review of laser point-scanning fluorescence microscopic techniques for their future developments and trends. In this article, the basic principles and recent advances in different point-scanning fluorescence microscopy imaging techniques are introduced from the perspectives of temporal resolution and spatial resolution, and the progress and applications of high spatio-temporal resolution microscopic imaging techniques based on point-scanning mode are summarized. Finally, the development trends and challenges of high spatio-temporal resolution point scanning fluorescence microscopic imaging technique are discussed.

Keywords:

Authors and contacts

1.
South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006, China
2.
School of Physics, South China Normal University, Guangzhou 510006, China

Corresponding author: Wang Bao-Ju, baoju.wang@m.scnu.edu.cn ; Zhan Qiu-Qiang, zhanqiuqiang@m.scnu.edu.cn

Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 62335008, 62122028, 11974123, 62105106), the Basic and Applied Basic Research Foundation of Guangdong Province, China (Grant Nos. 2023B1515040018, 2022A1515011395), the Basic and Applied Basic Research Foundation of Guangzhou, China (Grant No. 202201010376), and the China Postdoctoral Science Foundation (Grant Nos. 2021M691089, 2023T160237).

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Cited By

图 1 宽场和共聚焦显微成像技术对比　(a) 宽场显微镜系统简化图; (b) 宽场显微镜荧光信号采集方式^[18]; (c) 共聚焦显微镜系统简化图; (d) 共聚焦显微镜荧光信号采集方式^[18]; (e) 二维切面图对比^[18]; (f) 三维重构图对比^[18]

Figure 1. Comparison of wide-field and confocal microscopy: (a) Simplified system schematic of wide-field microscopy; (b) fluorescent signal acquisition of wide-field microscopy^[18]; (c) simplified system schematic of confocal microscopy; (d) fluorescent signal acquisition of confocal microscopy^[18]; (e) comparison of two-dimensional section images^[18]; (f) comparison of three-dimensional reconstruction images^[18].

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图 2 基于多焦点阵列的快速成像技术示意图 (a), (b) 像素重定位原理^[24,25]; (c) 共聚焦显微镜和ISM对100 nm直径荧光球的成像对比^[21]; (d) MSIM照明系统^[22]; (e) MSIM和宽场显微镜的活细胞双色成像对比^[22]; (f) MSIM的活细胞三维成像^[22]

Figure 2. Schematic diagram of fast imaging technology based on multifocus array: (a), (b) Principle of pixel reassignment^[24,25]; (c) imaging comparison of confocal microscope and ISM on 100 nm diameter fluorescent spheres^[21]; (d) illumination system for MSIM^[22]; (e) dual-color imaging comparison of MSIM and wide-field microscope on live-cell^[22]; (f) 3D imaging of live cells by MSIM^[22].

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图 3 基于微透镜阵列的快速成像技术示意图　(a) 转盘共聚焦系统简化图; (b) 实现Instant-SIM的关键步骤^[32]; (c) Instant-SIM和转盘共聚焦显微镜的活细胞双色成像对比^[32]

Figure 3. Schematic diagram of fast imaging technology based on microlens array: (a) Simplified schematic of the spinning disk confocal system; (b) key steps in implementing Instant-SIM^[32]; (c) dual-color imaging comparison of Instant-SIM and spinning disk confocal microscope on live-cell^[32].

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图 4 点扫描超分辨成像技术示意图　(a) STED原理图; (b) STED在NV色心上实现2.4 nm分辨率^[37]; (c) FED原理图^[43]; (d) 单颗粒FED成像^[44]; (e) RESOLFT原理图^[45]; (f)共聚焦显微镜和RESOLFT的细胞成像对比^[46]

Figure 4. Schematic diagram of point scanning super-resolution imaging technology: (a) STED schematic; (b) STED achieves 2.4 nm resolution on NV point^[37]; (c) FED schematic^[43]; (d) UCNPs-FED single particle imaging^[44]; (e) RESOLFT schematic^[45]; (f) cell imaging comparison of confocal microscope and RESOLFT^[46].

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图 5 上转换超分辨成像技术示意图　(a) 交叉弛豫传能荧光损耗机制^[57]; (b) 上转换STED超分辨成像结果^[57]; (c) 表面迁移荧光损耗机制^[60]; (d) SMED超分辨成像结果^[60]; (e) Yb³⁺/Pr³⁺共掺杂纳米颗粒的光子雪崩机制^[61]; (f) 光子雪崩超分辨成像结果^[61]

Figure 5. Schematic diagram of up-conversion super-resolution imaging technology: (a) Cross-relaxation energy transfer fluorescence loss mechanism^[57]; (b) up-conversion STED super-resolution imaging results^[57]; (c) surface migration fluorescence loss mechanism^[60]; (d) SMED super-resolution imaging results^[60]; (e) photon avalanche mechanism of Yb³⁺/Pr³⁺ co-doped nanoparticles^[61]; (f) photon avalanche super-resolution Resolution imaging results^[61].

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图 6 点扫描三维超分辨成像技术示意图　(a) 3D-STED PSF xz平面强度分布; (b) 3D-STED系统简化图^[67]; (c) 单SLM实现AO-3DSTED 装置^[69]; (d) 共聚焦显微镜和3D-STED对20 nm直径荧光球的成像对比^[67]; (e) AO-isoSTED系统简化图以及PSF xz平面强度分布^[73]; (f) 共聚焦显微镜和AO-isoSTED对细胞微管的成像对比^[73]

Figure 6. Schematic diagram of point scanning 3D super-resolution imaging technology: (a) 3D-STED PSF xz plane intensity distribution; (b) simplified schematic of 3D-STED system^[67]; (c) AO-3DSTED with a single SLM^[69]; (d) imaging comparison of confocal microscope and 3D-STED on 20 nm diameter fluorescent spheres^[67]; (e) simplified schematic of AO-isoSTED system and PSF xz plane intensity distribution^[73]; (f) imaging comparison of confocal microscope and AO-isoSTED on cell microtubules^[73].

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图 7 智能扫描超分辨成像技术示意图　(a) 智能RESOLFT扫描机制^[75]; (b) etSTED实验方案^[76]

Figure 7. Schematic diagram of intelligent scanning super-resolution imaging technology: (a) Smart RESOLFT scanning mechanism^[75]; (b) etSTED experimental scheme^[76].

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图 8 并行扫描超分辨成像技术示意图　(a) pRESOLFT 系统简化图^[71]; (b) pRESOLFT “ON” 状态下不同 I/I_s 的二维强度分布图^[71]; (c) pRESOLFT 纳米显微镜的活细胞成像^[71]; (d) 3D-pRESOLFT 三种模式的驻波叠加产生蜂窝状照明阵列^[81]; (e) 3D-pRESOLFT 纳米显微镜的活细胞成像^[81]

Figure 8. Schematic diagram of parallel scanning super-resolution imaging technology: (a) Simplified schematic of pRESOLFT system^[71]; (b) 2D profiles of the on-state probability distribution for different I/I_s ^[71]; (c) live-cell imaging with pRESOLFT nanoscopy^[71]; (d) three patterns of standing waves are superimposed to create a honeycomb lighting array in 3D-pRESOLFT^[81]; (e) live-cell imaging with 3D-pRESOLFT nanoscopy^[81].

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图 9 深度学习助力超高时空分辨率成像示意图　(a) 深度学习提高图像分辨率; (b) GAN网络训练结果^[82]; (c) RCAN网络训练结果^[84]; (d) Unet-RCAN网络训练结果^[85]

Figure 9. Schematic diagram of deep learning-assisted ultra-high spatial-temporal resolution imaging: (a) Deep learning improves image resolution; (b) GAN network training results^[82]; (c) RCAN network training results^[84]; (d) Unet-RCAN network training results^[85]

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表 1 SMLM, SIM, STED超分辨技术的关键性能指标对比

Table 1. Technical comparison of SMLM, SIM, STED super-resolution microscopy.

技术	横向分辨率/nm	轴向分辨率/nm	二维时间分辨率	激光类型	激光强度/ (W·cm^–2)	激光波段	荧光漂白程度	重构算法
SMLM^[3,4,7,8]	10—30	40—70	1—10 min (20 μm×20 μm)	CW	~1000	可见光	☆☆☆	需要
SIM^[10,11]	90—110	~300	1—10 ms (40 μm×40 μm)	CW	1—100	可见光	☆☆	需要
STED^[6,12,13]	20—50	40—150	1—20 s (50 μm×50 μm)	fs/ps	10⁹—10¹⁰	可见光	☆☆☆☆☆	无需

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表 2 不同点扫描超分辨成像技术的关键性能指标对比

Table 2. Overview of point-scanning super-resolution fluorescence microscopy techniques.

技术	横向分辨率 /nm	轴向分辨率 /nm	二维时间分辨率	激光类型	激光强度	荧光漂白程度	激光波段	组织成像深度	重构算法
Confocal^[19]	200	500	0.2—2.0 s (50 μm×50 μm)	CW	40 μW—1 mW	☆☆☆	可见光	中等	无需
Airyscan^[20]	120—140	400	0.2—1.0 s (50 μm×50 μm)	CW	4—20 μW	☆☆	可见光	低	需要
MSIM ^[22]	145	400	1 s (45.6 μm×45.6 μm)	CW fs/ps	1—25 μW 1.1 W	☆☆	可见光/ 近红外	低	需要
STED^[6,12,13]	20—50	40—150	13 s (50 μm×50 μm)	fs/ps	1—10 GW/cm²	☆☆☆☆☆	可见光	中等	无需
UCNPs-STED (SMED^[60])	17	～45	10—50 s (50 μm×50 μm)	CW	18 kW/cm²	零漂白	近红外	大	无需
RESOFLT^[50,52]	～40	～120	100 s (10 μm×10 μm)	fs/ps	1 kW/cm²	☆☆	可见光	中等	无需
pRESOFLT^[71,81]	～80	～80	0.4 s (10 μm×10 μm)	fs/ps	1 kW/cm²	☆☆	可见光	很低	需要
isoSTED^[70,73]	～40	～40	0.5—5.0 s (50 μm×50 μm)	fs/ps	50—100 mW	☆☆☆☆☆	可见光	中等	无需
MINFLUX^[53,54]	1—3	1—3	1—2 min (20 μm×20 μm)	CW	10—50 kW/cm²	☆	可见光	很低	需要
MINSTED^[55]	0.23	—	68 min (1.37 μm×1.37 μm)	fs/ps	1.5 μW	☆	可见光	很低	需要

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