Abstract
Methods and applications of light microscopy in the life sciences are compared with respect to 3D imaging, resolution, light exposure, sensitivity, and recording time. While conventional wide-field or laser scanning microscopy appear appropriate for smaller samples of only a few micrometers in size with a limited number of light exposures, light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or small organisms if multiple exposures or long measuring periods are desired. Super-resolution techniques should be considered in the context of high light exposure possibly causing photobleaching and photo-toxicity to living specimens.
Highlights
Wide-field as well as laser scanning methods can be combined with further techniques, e.g., Spectral Imaging [38,39,40] or Fluorescence Lifetime Imaging Microscopy (FLIM) [41,42,43,44,45], in order to distinguish between various fluorophores, molecular conformations, or cellular binding sites on the basis of their different fluorescence spectra or lifetimes
This manuscript gives an overview of methods of optical microscopy and their potential applications in the life sciences
Conventional wide-field microscopy or laser scanning microscopy appear appropriate for smaller samples (100 nm ≤ d ≤ 50 μm) with a limited number of light exposures, whereas light sheet microscopy appears to be an optimal method for larger 3D cell cultures, biopsies, or organisms (20 μm ≤ d ≤ 1 mm), if multiple exposures or long measuring periods are desired
Summary
Optical microscopy is a rapidly developing and widely used technique in life sciences such as biochemistry, biophysics, toxicology, genetics, and immunology. It is subdivided into two principal fields: Wide-field and laser scanning microscopy (LSM) [1,2]. Current techniques and applications are focused on 3D microscopy of the samples whose size exceeds the focal depth of a microscope objective lens, as well as on high resolution, contrast enhancement, and low light exposure, to avoid the photobleaching and photo-toxicity of living specimens. Methods are subdivided into transmission, scattering, and fluorescence microscopy, with fluorescence microscopy playing a predominant role in the life sciences. Clinical applications of an operation microscope, are not considered in this manuscript
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