自适应光学在双光子显微成像技术中的应用

Application of Adaptive Optics in Two⁃photon Microscopic Imaging

光学显微镜是生物医学研究必不可少的工具,其中双光子显微成像技术具有大深度三维显微成像功能,被认为是深层生物组织研究的首选工具。但是,在双光子成像系统使用过程中,光学系统的装配偏差、光学元件不理想以及生物样品的不均匀性都会在成像过程中引入像差,从而降低成像质量。通过在双光子显微成像系统中引入自适应光学技术,可实现对像差的有效校正,从而提高成像的分辨率、深度和视场。介绍了双光子显微成像中的像差来源和特点,概述了自适应光学技术中不同的探测和校正方法,综述了近年来自适应光学技术在双光子显微成像中不同的应用成果,最后对自适应光学在双光子显微成像中的发展进行了展望。

Significance Twophoton microscopy(TPM)has been widely used in biological imaging owing to its submicron lateral resolution,intrinsic optical sectioning,and deep penetration abilities.TPM enables the observation of cellular and subcellular dynamics in deep live tissues within highly complex and heterogeneous environments such as the mammalian brain,thereby providing critical in situ and in vivo information.However,because of the nonuniformity of the refractive index of biological tissues,the laser is distorted and scattered during propagation.Consequently,the focus point becomes a diffuse spot,which leads to a decreased imaging depth and poor resolution of TPM.Adaptive optics(AO)technology was first applied to TPM in 2000,where a genetic algorithm was used to calculate the wavefront distortion and a deformable mirror(DM)was used to correct the aberration introduced by biological samples.Since then,various AO schemes have been developed for a wide range of highresolution microscopes to advance the development of biological exploration.In this study,the sources and characteristics of aberrations in TPM are examined,and different detection and correction methods in AO are summarized.The different applications of AO in TPM in recent years are comprehensively reviewed.Progress For AO technology,wavefront detection methods are generally divided into direct wavefront detection,which uses a wavefront sensor(WS)to detect wavefronts,and indirect wavefront detection,which estimates the aberrated wavefront using iterative algorithms.In 2010,the pupilsegmentation AO method was proposed by Ji et al.This method involves the division of the pupil into several sub apertures and the use of spatial light modulator(SLM)to modulate the wavefront phase,and the imaging resolution of a fixed mouse cortex slice was restored to the neardiffractionlimited(Fig.2).In 2012,Tang et al.proposed an iterative multiphoton adaptive compensation technique that exploits the nonlinearity of multiphoton signals to determine and compensate for distortions and focus light inside deep tissues.The technique was tested using a variety of highly heterogeneous biological samples,and an imaging resolution of approximately 100 nm was obtained(Fig.3).In 2014,Wang et al.adopted a digital micromirror device(DMD)to rapidly modulate the intensity or phase of light rays of multiple pupil segments in parallel to determine the wavefront aberration(Fig.4).Subsequently,Park et al.developed a multipupil adaptive optics(MPAO)method in 2017 that allows the simultaneous correction of a wavefront over a field of view of 450µm×450µm,thereby expanding the correction area to nine times larger than those of conventional methods(Fig.7).Recently,Rodríguez et al.developed a compact adaptive optics module and incorporated it into both TPM and threephoton microscopy to correct tissueinduced aberrations.They also demonstrated that their technology allows the in vivo highresolution imaging of both neuronal structures and somatosensoryevoked calcium responses in the spinal cord of mice at great depths(Fig.5).Generally,the indirect wavefront detection system is relatively simple and easy to implement,but it is also timeconsuming and has high computational expense.In the typical TPM,the excited fluorescence is limited to a small area near the focus.Such fluorescence points act as a natural guide star for the wavefront sensor to allow the application of direct wavefront detection in TPM.From 2010 to 2013,Cha et al.and Tao et al.used SharkHartmann wavefront sensor(SHWS)to detect the emission light and DM to correct the excitation light by injecting foreign fluorescent substances into the sample as a guide star for the in vitro imaging of mice brain tissue(Fig.10).In 2014,Wang et al.proposed descanning technology to accumulate all the transmitted optical signals for wavefront detection and improve the quality and efficiency of direct wavefront detection.The SLM was used to correct the excitation light and in vivo structural imaging was performed on the brain neurons of mice(Fig.11).In 2019,Liu et al.used nearinfrared fluorescent dye as a guide star for direct wavefront detection,but replaced the SLM with DM.Structural imaging was carried out on the microvessels and neurons of mice,and an imaging depth of up to 1100µm was obtained(Fig.12).Compared with indirect wavefront detection,direct wavefront detection is faster and more accurate.However,the optical system for direct wavefront detection is complex,which reduces the imaging signaltonoise ratio.To effectively use the two detection methods,the real distorted wavefront should be obtained in the optical detection path without a wavefront sensor.In 2017,Papadopoulos et al.proposed the focus scanning holographic aberration probing(FSHARP)method,which directly measures the point spread function(PSF)of the distorted wavefront using interference technology and corrects the wavefront using the phase conjugation of the PSF(Fig.14).In 2022,Qu et al.combined a conjugate AO with the FSHARP system and used a phaselocked amplifier to simplify the measurement steps of PSFs,which improves the measurement speed and correction accuracy(Fig.15).Conclusions and Prospects Currently,there is an increasing demand for highresolution structural and functional neuroimaging systems owing to the rapid development of brain science.However,the aberrations caused by tissues are complex,irregular,and rapidly changing and fast aberration detection and accurate correction systems are required.Therefore,it is necessary to use highperformance adaptive elements such as a high sensitivity wavefront sensor and correction elements with a high refresh rate and large compensation range.In addition,fast and accurate compensation algorithms can also improve the AO performance.In summary,the effective combination of various detection,correction and control techniques is the focus of in vivo microscopic imaging,which provides valuable information for scientific research.