Extending the Resolving Power of the Light Microscope in Time and Space

Abstract

The conventional widefield light microscope, whether it is brightfield, darkfield, or fluorescence, is simply an optical bench that is set up so that objects can be comfortably observed by the eyes. Improvement in resolution beyond the diffraction limit of widefield or confocal microscopy is superresolution microscopy, or nanoscopy. Expansion microscopy, developed in 2015, increases the resolving power of the system by physically expanding a digested tissue sample uniformly. Light sheet fluorescence microscopy uses a sheet of light to illuminate the entire lateral widefield view. Illumination of the entrance pupil of the excitatory lens with different beam structures can produce very thin light sheets. Total internal reflection fluorescence microscopy generates an evanescent wave of light energy about 100 nm into plasma membrane and cytoplasm of the cell, which have a lower refractive index than the medium through which the incident light is traveling. Directly imaging fluorescence lifetimes in real space uses a streak camera.