Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Elisa DʼEste; Gražvydas Lukinavičius; Richard Lincoln; Felipe Opazo; Eugenio F Fornasiero

doi:10.1016/j.tcb.2023.12.001

Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Trends Cell Biol. 2024 Jan 5:S0962-8924(23)00239-8. doi: 10.1016/j.tcb.2023.12.001. Online ahead of print.

Authors

Elisa DʼEste¹, Gražvydas Lukinavičius², Richard Lincoln³, Felipe Opazo⁴, Eugenio F Fornasiero⁵

Affiliations

¹ Optical Microscopy Facility, Max Planck Institute for Medical Research, Heidelberg 69120, Germany. Electronic address: elisa.deste@mr.mpg.de.
² Chromatin Labelling and Imaging Group, Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Göttingen 37077, Germany. Electronic address: grazvydas.lukinavicius@mpinat.mpg.de.
³ Department of Optical Nanoscopy, Max Planck Institute for Medical Research, Heidelberg 69120, Germany. Electronic address: richard.lincoln@mr.mpg.de.
⁴ Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen (UMG), Göttingen 37073, Germany; Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center, Göttingen 37075, Germany; NanoTag Biotechnologies GmbH, Göttingen 37079, Germany. Electronic address: fopazo@gwdg.de.
⁵ Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen (UMG), Göttingen 37073, Germany; Department of Life Sciences, University of Trieste, Trieste 34127, Italy. Electronic address: efornas@gwdg.de.

PMID: 38184400
DOI: 10.1016/j.tcb.2023.12.001

Abstract

Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications.

Keywords: labeling; nanoscale imaging; sample preparation; super-resolution microscopy.

Publication types

Review