多色单分子定位显微技术研究进展(特邀)

Advances in Multicolor Single-molecule Localization Microscopy(Invited)

超分辨显微镜是探测分子尺度的亚细胞结构的有力工具,为生物学研究提供了新的途径。由于许多生物学问题都可以通过分析不同细胞结构间的相互作用进行研究,因此近年来发展了一系列多色超分辨显微技术。本文从生物样品制备和光学系统改进两个角度,对已有的多色单分子定位显微技术进行了总结,简要概括了每种技术的基本原理,分析了每种技术的优缺点及适用范围,重点归纳了近5年通过光学系统改进提升多色单分子定位显微技术性能的各类方法。最后,对快速发展的多色单分子定位显微技术领域进行了展望。

Due to its simplicity and minimal invasion,light microscopy has been widely applied in biological science,medicine and chemistry.However,as the result of diffraction,the spatial resolution of standard optical microscopy is limited to about half the wavelength of light,with a lateral dimension of around 200~300 nm and an axial dimension of approximately 500~700 nm.This diffraction limits the ability of light microscopy to resolve the subcellular organization of individual molecules or molecular complexes.To overcome the resolution barrier of optical microscopy caused by diffraction limit,a variety of superresolution microscopy techniques have been developed over the past 15 years,which significantly improved the resolution of light microscopy to 10 nm.These techniques can be roughly classified into two categories:1)single-molecule localization microscopy such as stochastic optical reconstruction microscopy and photoactivation localization microscopy,and 2)spatially patterned illumination-based methods,such as stimulated emission depletion microscopy and structured illumination microscopy.These techniques have revolutionized our understanding of the nanoscale world,Bringing new opportunities and challenges to molecular-scale biological research.This review summarizes the multicolor single-molecule localization microscopy techniques developed in the past five years from the perspectives of biological sample preparation and optical system improvement,The basic principles of each technique are explained and their advantages and disadvantages are discussed.According to the type of fluorescent probes select during sample preparation,some multicolor SMLM can be classified into three categories:Activator-free SMLM technology using synthetic dyes,which requires fluorophores that exhibit similar blinking efficiency under identical photoswitching buffer conditions.techniques using probes with both an activator and a reporter fluorophore enable multicolor SMLM using different activation lasers.And spectrally selective activation of the reporter fluorophores and multicolor SMLM by repetitive quenching and labelling biological samples.The addition of optical elements allows different wavelengths to be processed according to the spectral information of a fluorophore and multicolor imaging can be realized through data post-processing.These multicolor SMLM methods are mainly divided into three categories:Ratiometric SMLM,spectrally resolved SMLM and methods based on Point-Spread-Function(PSF)engineering.In Ratiometric SMLM,synthetic dyes can be efficiently excited by the same laser wavelength in the same switching buffer,but exhibit different emission peaks.The emission fluorescence is spectrally separated by a dichroic beam splitter and imaged into two parts of the same camera.Spectrally resolved SMLM separate the emission fluorescence of different dyes through a dispersive device,and records the emission spectrum and position information separately.The last method for multicolor single-molecule localization is PSF engineering.Additional optical elements are used to make the PSF shape vary with the depth of the emitter and the wavelength of the fluorophore.The problem with the Ratiometric approach is that the Crosstalk between channels is higher than in the other two methods.However,an outstanding challenge of the last two techniques is the accurate and precise localization of individual point emitters in densely labelled samples.Finally,we present an outlook on the fast-growing field of multicolor single-molecule localization microscopy.One of the future development directions of multicolor single molecules is the research on probes,including the brighter organic fluorescent dyes or fluorescent proteins and more universal imaging buffers.Further research can also be done on reducing the complexity of the optical system and expanding the field of view of imaging.It can be envisioned that multicolor SMLM will play a more prominent role in biological and medical research for many years to come.We hope that this review can help researchers choose the appropriate multicolor SMLM for their own experiments.