Confocal Fluorescence Microscopy

Abstract

This article describes basic approaches to image materials by means of optical microscopy with fluorescence markers. The fluorescence microscopy is used to image materials in which the contrast cannot be achieved by light absorption, scattering, or birefringence. The fluorescence microscopy benefits greatly from a confocal mode of observation that eliminates signal coming from outside the small region of sample that is probed by a tightly focused laser beam. By scanning the focused laser beamthrough the sample, one obtains a 3D map of density of fluorescing molecules staining the sample. This technique is called confocal laser scanning microscopy (CLSM). By using fluorescent markers of anisometric shape and a polarized laser beam, one can produce 3D images of orientationally ordered materials such as liquid crystals. In this technique, called a fluorescent confocal polarizing microscopy (FCPM), the signal depends on the angle between the transition dipole of the dye and the polarization of probing light. Recent advances in the development of fluorescent markers that can be controllably switched between the bright state and the dark state lead to revolutionary development of far‐field optical microscopy with resolution well below the diffraction limit.

A link between mitotic defects and mitotic catastrophe: detection and cell fate

“…Confocal fluorescence microscopy as well as a wide range of fluorophores allow researchers to obtain clear images of different biological structures in different scanning planes and to detect micronucleation [79,80]. Thus, researchers have recommended various imaging approaches to detect mitotic catastrophe; however, confocal fluorescence microscopy is recommended to obtain convincing results.…”

Morphogenesis of defects and tactoids during isotropic–nematic phase transition in self-assembled lyotropic chromonic liquid crystals

“…We remind the reader that in regular optical polarizing microscopy, the image is determined by the integral over the (generally distorted) configuration of the optic axis along the pathway of light propagation. If the sample is thin, the undesirable director distortions along the light propagation direction are suppressed and the image represents a 2D pattern of the director field that can be reconstructed by using a microscope with an LC-PolScope universal compensator [61].…”

Selecting the best surface analysis method for your materials/samples

“…For the characterization of surfaces, reflective modalities, such as confocal reflectance microscopy, are most commonly employed. Lavrentovich provides a good introduction to confocal fluorescence microscopy including theory, 54 and Ribbe et al provide an early example of characterization of a thin film using this method. 55 Additional considerations/limitations: • Many atoms and molecules are not fluorescent, specifically designed dyes are often used to label chemical/biological moieties of interest.…”