Research and Prospect for Nanoscale Resolution Microscopy
Fluorescence microscopy is a vital tool in life science research,but the diffraction nature of light limits further observation of cells.Super-resolution imaging techniques provide deeper insights into cellular structures,including stimulated emission depletion microscopy(STED),structured illumination microscopy(SIM),and single-molecule localization microscopy(SMLM).Each of these methods offers unique advantages and principles that push the boundaries of spatial resolution beyond conventional diffraction limits.Among these techniques,SMLM stands out for its exceptional resolution,offering nanometer resolution and becoming a powerful tool for obtaining high-resolution images.SMLM is particularly valuable for studying the spatial distribution and interactions of organelles and macromolecular complexes.Following the award of the Nobel Prize in Chemistry in 2014,super-duper resolution imaging techniques were listed as one of Nature's seven technologies to watch in 2024.The development of these techniques remains an important area of research.We introduce the development of multi-color SMLM,three-dimensional(3D)SMLM,and nanoscale resolution microscopes.We describe several methods to achieve multi-color SMLM.Sequential imaging and Exchange-PAINT require image targets in sequence,excitation or emission spectral demixing can obtain multi-color images simultaneously based on spectral difference between fluorescent dyes,dual-channel spectroscopic SMLM to achieve simultaneous imaging and spectral analysis of each molecule,and techniques based on binding kinetics of PAINT achieve multi-color by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT.We then discuss various approaches for 3D imaging.Point spread function(PSF)engineering techniques manipulate the shape and properties of the PSF to improve 3D localization accuracy.Multi-plane imaging methods capture images from different focal planes and reconstruct them to obtain 3D information.Interferometry methods use single molecule interference to achieve high precision in axial localization,providing another way for high resolution 3D nanoscopy.Finally,we highlight advances in new nanoscale resolution microscopes based on modulated illumination patterns,including minimal photon fluxes(MINFLUX),repetitive optical selective exposure(ROSE),ROSE-Z,SIMFLUX,SIMPLE,and ModLoc.MINFLUX is known for its ability to achieve ultra-high resolution by detecting minimal photon fluxes from single molecules using a doughnut-shaped excitation spot to spatially modulate excitation intensities.Typically,we focus on ROSE and ROSE-Z,which outperform other techniques,using a resonant mirror to eliminate localization errors caused by fluorescence blinking.Recently,resolution enhancement by sequential imaging(RESI)and one nanometre expansion(ONE)was introduced to achieve resolution down to the Ångström scale.Nanoscopy serves as a new role between super resolution microscopy and structural biology and will lead to more discoveries in complex biological systems.Overall,this article provides a comprehensive overview of current advances in super-resolution imaging techniques,highlighting their contributions to overcoming the diffraction limit and enabling detailed observation of nanoscale biological structures,and provides an outlook on promising new techniques and applications.Through detailed descriptions of the principles,benefits,and applications of multi-color and 3D techniques,the article highlights new nanoscale imaging techniques that are expanding our ability to visualize and understand the intricate details of molecular and cellular processes.We hope that this article can be a primer resource for both newcomers and seasoned practitioners of SMLM.
super resolution microscopysingle molecule localization microscopymulti-color imaging3D imagingnanoscopy
万方数据
维普
