间接波前整形近红外二区共聚焦系统及 活体组织成像研究

荧光成像技术具有高分辨率、高灵敏性、非侵入性、和特异性标记等特点，因此被广泛应用于生物成像领域。激光扫描共聚焦显微镜具有光学切片能力和分辨率高的特点，可以获得生物组织丰富的三维结构信息。然而，因为光子在生物组织的强烈吸收和散射作用，光学成像技术在深层组织成像领域特别是脑组织成像领域的应用受到限制。尽管借助近红外二区波段高组织穿透的光学特性可以突破限制进行组织成像，但在深层组织成像时图像质量仍然受到散射影响。通过波前整形技术，可以对由生物组织不均匀性所导致的多次光散射进行相位补偿，提升光束在生物组织内的聚焦能力。因此，本研究使用间接波前整形技术，提升近红外二区共聚焦成像系统在活体组织成像的能力。

本研究设计并搭建了一台近红外二区激光扫描共聚焦显微成像系统，并且结合了波前整形技术，通过光路设计具有两种成像模式且可以轻易切换，之后对小鼠进行鼠耳和脑部血管结构活体成像。成像系统使用特定的光学元件，响应波段覆盖全部传统近红外二区（900-1700 nm），使用808 nm激发光源，实现了近红外二区高分辨率三维层析成像，达到了横向570 nm和轴向1.7 μm的空间分辨率，接近理论极限。使用成像系统对小鼠脑组织进行近红外二区活体成像，在不进行开颅手术和透明化处理的条件下，获得300 μm深度的血管网络结构。设计并编写波前整形优化程序，通过空间光调制器对入射光进行调控，让成像系统可以在不增加额外传感器和引导星的情况下进行波前整形相位优化。在体外实验中，波前整形优化程序可以使图像的荧光信号强度获得4倍提升，并且在轴向漂移导致的离焦和加入散射介质的成像条件下都展现出校正和优化效果。活体成像实验中，在相同的成像条件下，通过波前整形优化获得更多的生物组织深层信息。本研究使用波前整形技术，通过相位调控的方法，提升对活体生物组织的近红外二区成像能力，有望为生物组织深层成像提供新的策略。

Fluorescence imaging technology has widespread application in the field of biological imaging due to its characteristics of high resolution, high sensitivity, non-invasiveness, non-radiation and specific labeling. Laser scanning confocal microscopy has the characteristics of optical slicing capability and high resolution, which can obtain sufficient 3D structure information of biological tissues. However, the strong absorption and scattering of photons by biological tissues limit its application in deep tissue imaging, especially for brain tissue. Although the high tissue penetration properties offer the NIR-Ⅱ light offer potential for overcoming this limitation for deep tissue imaging, image quality is still affected by scattering in depth imaging. Via wavefront shaping technology, phase compensation can be implemented to mitigate the effects of multiple light scattering induced by non-uniformity in biological tissues, thereby enhancing the beam focusing capability within biological samples. Therefore, this study uses indirect wavefront shaping technology to enhance the imaging ability of NIR-Ⅱ confocal imaging system in vivo tissue imaging.

In this study, we designed and built a NIR-Ⅱ laser scanning confocal microscopy imaging system combined with wavefront shaping technology, which has two imaging modes and can be easily switched through the optical path design. Afterwards, we performed in vivo imaging of the mouse ear and brain vascular structures. The imaging system uses specific optical elements, which ensure the response band covers the total traditional NIR-Ⅱ (900-1700 nm). With the excitation of 808 nm laser, we achieved the high-resolution NIR-Ⅱ 3D tomography, with a 570 nm lateral resolution and a 1.7 μm axial resolution, which are close to the theoretical limits. Using the microscope, we imaged the mouse brain tissue in vivo, obtaining vascular network structures up to 300 μm in depth without craniotomy or skull hyalinization. We designed and programmed a wavefront shaping optimization program, enabling control of incident light through a spatial light modulator for phase optimization without additional phase sensors or guide stars. In the vitro experiment, the wavefront shaping optimization program can improve the fluorescence signal intensity of the image by 4 times, it also shows the correction and optimization effects under the defocus caused by axial drift and the imaging conditions with the addition of scattering medium. For in vivo imaging, we obtained more information of deep tissue via wavefront shaping optimization with the same imaging conditions. This study is using wavefront shaping to improve the imaging ability of biological tissues through phase regulation, which may provide a new strategy for deep imaging of biological tissues.

[

[1] SCHOUW H M, HUISMAN L A, JANSSEN Y F, et al. Targeted optical fluorescence imaging: a meta-narrative review and future perspectives[J]. European Journal of Nuclear Medicine and Molecular Imaging, 2021: 1-21.

[2] 蔡朝冲. 近红外Ⅱ区荧光高分辨活体成像研究[D]. 浙江大学, 2020.

[3] MINSKY M. Microscopy apparatus US patent 3013467[J]. USP Office, Ed. US, 1961, 658.

[4] ELLIOTT A D. Confocal microscopy: principles and modern practices[J]. Current Protocols in Cytometry, 2020, 92(1): e68.

[5] XI P, RAJWA B, JONES J T, et al. The design and construction of a cost-efficient confocal laser scanning microscope[J]. American Journal of Physics, 2007, 75(3): 203-207.

[6] ZHANG Y, HU B, DAI Y, et al. A new multichannel spectral imaging laser scanning confocal microscope[J]. Computational and Mathematical Methods in Medicine, 2013, 2013.

[7] WILSON T. Resolution and optical sectioning in the confocal microscope[J]. Journal of Microscopy, 2011, 244(2): 113-121.

[8] LI J, FENG Z, YU X, et al. Aggregation-induced emission fluorophores towards the second near-infrared optical windows with suppressed imaging background[J]. Coordination Chemistry Reviews, 2022, 472: 214792.

[9] SMITH A M, MANCINI M C, NIE S. Second window for in vivo imaging[J]. Nature Uanotechnology, 2009, 4(11): 710-711.

[10] HONG G, ANTARIS A L, DAI H. Near-infrared fluorophores for biomedical imaging[J]. Nature Biomedical Engineering, 2017, 1(1): 0010.

[11] FENG Z, TANG T, WU T, et al. Perfecting and extending the near-infrared imaging window[J]. Light: Science & Applications, 2021, 10(1): 197.

[12] DIAO S, HONG G, ANTARIS A L, et al. Biological imaging without autofluorescence in the second near-infrared region[J]. Nano Research, 2015, 8: 3027-3034.

[13] WANG T, CHEN Y, WANG B, et al. Recent progress of second near-infrared (NIR-II) fluorescence microscopy in bioimaging[J]. Frontiers in Physiology, 2023, 14: 1126805.

[14] MENG X, PANG X, ZHANG K, et al. Recent Advances in Near‐Infrared‐II Fluorescence Imaging for Deep‐Tissue Molecular Analysis and Cancer Diagnosis[J]. Small, 2022, 18(31): 2202035.

[15] POTMA E O, KNEZ D, ETTENBERG M, et al. High-speed 2D and 3D mid-IR imaging with an InGaAs camera[J]. APL Photonics, 2021, 6(9).

[16] LIU Z, ZHU Y, ZHANG L, et al. Structural and functional imaging of brains[J]. Science China Chemistry, 2023, 66(2): 324-366.

[17] BAGHDASARYAN A, WANG F, REN F, et al. Phosphorylcholine-conjugated gold-molecular clusters improve signal for Lymph Node NIR-II fluorescence imaging in preclinical cancer models[J]. Nature Communications, 2022, 13(1): 5613.

[18] XU Y, LI C, MA X, et al. Long wavelength–emissive Ru (II) metallacycle–based photosensitizer assisting in vivo bacterial diagnosis and antibacterial treatment[J]. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(32): e2209904119.

[19] NIU X, WEI P, SUN J, et al. Biomineralized hybrid nanodots for tumor therapy via NIR-II fluorescence and photothermal imaging[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 1052014.

[20] WELSHER K, LIU Z, SHERLOCK S P, et al. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice[J]. Nature Nanotechnology, 2009, 4(11): 773-780.

[21] BRUNS O T, BISCHOF T S, HARRIS D K, et al. Next-generation in vivo optical imaging with short-wave infrared quantum dots[J]. Nature Biomedical Engineering, 2017, 1(4): 0056.

[22] SUO Y, WU F, XU P, et al. NIR‐II fluorescence endoscopy for targeted imaging of colorectal cancer[J]. Advanced Healthcare Materials, 2019, 8(23): 1900974.

[23] ZHENG Z, JIA Z, QU C, et al. Biodegradable silica‐based nanotheranostics for precise MRI/NIR‐II fluorescence imaging and self‐reinforcing antitumor therapy[J]. Small, 2021, 17(10): 2006508.

[24] LI J, JIANG R, WANG Q, et al. Semiconducting polymer nanotheranostics for NIR-II/Photoacoustic imaging-guided photothermal initiated nitric oxide/photothermal therapy[J]. Biomaterials, 2019, 217: 119304.

[25] ZHOU W, YIN L, ZHANG X, et al. Recent advances in small molecule dye-based nanotheranostics for NIR-II photoacoustic imaging-guided cancer therapy[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 1002006.

[26] HAN B, HU X, ZHANG X, et al. The fluorescence mechanism of carbon dots based on the separation and identification of small molecular fluorophores[J]. RSC Advances, 2022, 12(19): 11640-11648.

[27] KASPRZYK W, ŚWIERGOSZ T, ROMAŃCZYK P P, et al. The role of molecular fluorophores in the photoluminescence of carbon dots derived from citric acid: current state-of-the-art and future perspectives[J]. Nanoscale, 2022, 14(39): 14368-14384.

[28] RODRÍGUEZ-LUNA M R, OKAMOTO N, AL-TAHER M, et al. In vivo imaging evaluation of fluorescence intensity at tail emission of near-infrared-I (NIR-I) fluorophores in a porcine model[J]. Life, 2022, 12(8): 1123.

[29] HE L, HE L H, XU S, et al. Engineering of reversible NIR‐II redox‐responsive fluorescent probes for imaging of inflammation in vivo[J]. Angewandte Chemie International Edition, 2022, 134(46): e202211409.

[30] QIN Z, REN T B, ZHOU H, et al. NIRII‐HDs: A Versatile Platform for Developing Activatable NIR‐II Fluorogenic Probes for Reliable In Vivo Analyte Sensing[J]. Angewandte Chemie International Edition, 2022, 61(19): e202201541.

[31] REN T B, WANG Z Y, XIANG Z, et al. A General Strategy for Development of Activatable NIR‐II Fluorescent Probes for In Vivo High‐Contrast Bioimaging[J]. Angewandte Chemie International Edition, 2021, 133(2): 813-818.

[32] GREINER J, SANKARANKUTTY A C, SEIDEL T, et al. Confocal microscopy-based estimation of intracellular conductivities in myocardium for modeling of the normal and infarcted heart[J]. Computers in Biology and Medicine, 2022, 146: 105579.

[33] OJHA S, PRIBYL J, KLIMOVIC S, et al. Measurement of liver stiffness using atomic force microscopy coupled with polarization microscopy[J]. JoVE (Journal of Visualized Experiments), 2022 (185): e63974.

[34] MIRSANAYE K, URIBE CASTAÑO L, KAMALIDDIN Y, et al. Unsupervised determination of lung tumor margin with widefield polarimetric second-harmonic generation microscopy[J]. Scientific Reports, 2022, 12(1): 20713.

[35] SONODA K, HARADA M, AOMURA D, et al. Relationship between glomerular number in fresh kidney biopsy samples and light microscopy samples[J]. Clinical and Experimental Nephrology, 2022, 26(5): 424-434.

[36] HANAFY B G, ABUMANDOUR M M A, KANDYLE R, et al. Ultrastructural characterization of the intestine of the Eurasian common moorhen using scanning electron microscopy and light microscopy[J]. Microscopy Research and Technique, 2022, 85(1): 106-116.

[37] TEO A W J, MANSOOR H, SIM N, et al. In vivo confocal microscopy evaluation in patients with keratoconus[J]. Journal of Clinical Medicine, 2022, 11(2): 393.

[38] HE M, WU D, ZHANG Y, et al. Protein-enhanced NIR-IIb emission of indocyanine green for functional bioimaging[J]. ACS Applied Bio Materials, 2020, 3(12): 9126-9134.

[39] HE M, LI D, ZHENG Z, et al. Aggregation-induced emission nanoprobe assisted ultra-deep through-skull three-photon mouse brain imaging[J]. Nano Today, 2022, 45: 101536.

[40] ZHANG M, YUE J, CUI R, et al. Bright quantum dots emitting at∼ 1,600 nm in the NIR-IIb window for deep tissue fluorescence imaging[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(26): 6590-6595.

[41] WANG F, WAN H, MA Z, et al. Light-sheet microscopy in the near-infrared II window[J]. Nature Methods, 2019, 16(6): 545-552.

[42] HAMPSON K M, TURCOTTE R, MILLER D T, et al. Adaptive optics for high-resolution imaging[J]. Nature Reviews Methods Primers, 2021, 1(1): 68.

[43] SKIPETROV S E. Langevin description of speckle dynamics in nonlinear disordered media[J]. Physical Review E, 2003, 67(1): 016601.

[44] RODRÍGUEZ C, CHEN A, RIVERA J A, et al. An adaptive optics module for deep tissue multiphoton imaging in vivo[J]. Nature Methods, 2021, 18(10): 1259-1264.

[45] CONKEY D B, CARAVACA-AGUIRRE A M, PIESTUN R. High-speed scattering medium characterization with application to focusing light through turbid media[J]. Optics Express, 2012, 20(2): 1733-1740.

[46] YOON S, CHEON S Y, PARK S, et al. Recent advances in optical imaging through deep tissue: Imaging probes and techniques[J]. Biomaterials Research, 2022, 26(1): 57.

[47] JI N, SATO T R, BETZIG E. Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(1): 22-27.

[48] TANG J, GERMAIN R N, CUI M. Superpenetration optical microscopy by iterative multiphoton adaptive compensation technique[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(22): 8434-8439.

[49] PARK J H, SUN W, CUI M. High-resolution in vivo imaging of mouse brain through the intact skull[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(30): 9236-9241.

[50] 谭天, 史天悦, 吴长锋, 等. 基于间接波前整形的近红外二区荧光共聚焦成像研究[J]. 中国光学 (中英文), 2024, 17(1): 150-159.

[51] LIU Y, LIU J, CHEN D, et al. Fluorination enhances NIR‐II fluorescence of polymer dots for quantitative brain tumor imaging[J]. Angewandte Chemie International Edition, 2020, 132(47): 21235-21243.

[52] BEHNKE T, WÜRTH C, HOFFMANN K, et al. Encapsulation of hydrophobic dyes in polystyrene micro-and nanoparticles via swelling procedures[J]. Journal of Fluorescence, 2011, 21: 937-944.

[53] WEBB R H. Confocal optical microscopy[J]. Reports on Progress in Physics, 1996, 59(3): 427.

[54] LIM C S, HEO J H, YOU M S, et al. Synthesis of PS-b-P2VP di-block copolymer particles with internal structure via simple reprecipitation method[J]. Macromolecular Research, 2014, 22: 324-328.

[55] LAMBORA A, GUPTA K, CHOPRA K. Genetic algorithm-A literature review[C]//2019 international conference on machine learning, big data, cloud and parallel computing (COMITCon). IEEE, 2019: 380-384.