Fluorescence Microscopy—An Outline of Hardware, Biological Handling, and Fluorophore Considerations

Abstract

Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability; (2) amenability of biological models to fluorescence microscopy; and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology.

Microscopic Visualization of Cell-Cell Adhesion Complexes at Micro and Nanoscale

“…For example, researchers visualized E-cadherin labeled cell-cell junctions in mouse pancreas using intravital microscopy combined with multiphoton imaging ( Erami et al, 2016 ). However, imaging in live animals has many technical challenges while it also requires access to high-resolution microscopy techniques (reviewed in ( Choi et al, 2015 ; Hickey et al, 2022 ). Smaller mammalian species might be considered, including larval zebrafish, because of their high optical transparency that enables direct visual access to biological processes ( Ahrens et al, 2012 ; Høgset et al, 2020 ).…”

“…Recent advances do include co-cultures of multiple cell types and researchers are increasingly using 3D cell models to recreate organized, miniaturized versions of organs to study diseases ( Martin 2020 ). All three types of imaging ways have their advantages and disadvantages as well as technical difficulties and necessary equipment (reviewed in ( Hickey et al, 2022 )).…”

“…The axial resolution of most light-based techniques is not enough when imaging deeper in the cells. Next to growing cells in 3D [like organoids ( Martin 2020 ; Hickey et al, 2022 )], imaging epithelial or endothelial tissue sections is another good approach. Tissue sections have the major advantage of having preserved complex interactions among cells and their microenvironment and therefore cells kept their cell polarity in the hierarchical architecture of the tissue.…”

“…Most fluorescent imaging techniques, either confocal or super-resolution, allow for imaging on living cells/samples within a controlled environment and the possibility of time-lapse imaging for days. However, possible phototoxic events after long illumination, as seen in super-resolution techniques like STED and SIM, need to be taken into consideration [reviewed and solutions in ( Tosheva et al, 2020 ; Hickey et al, 2022 )].…”

“…Labels/probes need to bind the protein of interest correctly and these can be directly visualized or followed by targeting of an imaging label (in case of fluorescence microscopy) or gold particle (in case of EM). Therefore, applying appropriate controls when setting up an experiment is highly desirable [discussed in ( Hickey et al, 2022 )]. The easiest are techniques without the need for labelling to see subcellular structures, which include SXT and cryo-EM.…”