TABLE 1.

Comparison of light microscopy techniques based on their advantages and limitations.

Method	Tissue preparation	Spatial resolution	Depth	Main uses	In vivo imaging	Advantages	Limitations	References
Epifluorescence	-Fixation -In vivo: cranial window	350–500 nm	<200 μm	Rapid verifications (i.e. staining outcome, morphology, distribution)	Possible, but not typically used	-Easy use -Low cost -Rapid -Multi-channels -Optical sectioning not required	-Light diffraction limit -Out-of-focus background fluorescence detection	Yang and Yuste, 2017; Bayerl et al., 2019
Slide scanner microscopes (brightfield or fluorescent)	-Fixation -Slide- mounted	350–500 nm	<200 μm	-Cell distribution, density and morphology -Surface area -3D reconstructions (whole brain or serial sections)	No	-Easy use -High-speed scanning of large tissue sections -Multi-channel	-Light diffraction limit -Out-of-focus background fluorescence detection	Mikula et al., 2007; Chen et al., 2014; Yang and Yuste, 2017; Roetzer et al., 2019
Confocal	-Fixation -Tissue clearing	200–800 nm	<100 μm	-2D imaging of tissue sections -3D reconstructions (z stacks)	Possible, but not typically used	-Reduction of out of focus background fluorescence detection -Multi-channels	-Photobleaching -Slow speed: not suitable for following rapid changes in dynamic phenomena	Graf and Boppart, 2010; Pérez-Alvarez et al., 2013; Oreopoulos et al., 2014; Villaseñor and Collin, 2017
2-photon	-Fixation -In vivo: thinned-skull or cranial window -Chronic in vivo: canula insertion	400–900 nm	>300 μm >500 μm in vitro	-Live in vivo imaging -Time-lapse imaging -3D reconstructions	Yes	-Reduction of out of focus background fluorescence detection -Reduction of light scattering -Increased spatial and temporal resolution -Multi-channels	-Photobleaching (less than confocal microscopes) -Slightly decreased resolution due the use of larger wavelengths	Santi, 2011; Pérez-Alvarez et al., 2013; Oreopoulos et al., 2014
STED	-Fixation -In vivo: thinned-skull or cranial window -Chronic in vivo: canula insertion	65–100 nm	10–15 μm	-Synapse and dendritic spine dynamics	Yes	-Subcellular resolution -Multi-channels	-High cost and difficult equipment accessibility -Low tissue-penetration depth	Klar et al., 2001; Westphal et al., 2008; Berning et al., 2012; Chéreau et al., 2015
Light-sheet	-Fixation -Tissue clearing -Organotypic cultures	270–1000 nm	750 μm (live tissue) 2 mm (cleared tissue)	-Whole small organisms imaging (i.e. mouse embryos) or brain -Overview of cellular networks -3D reconstructions	No	-High-speed scanning of large tissue sections -Reduced photobleaching -High tissue-penetration depth	-Not suitable for large organisms such as adult mice (i.e. suitable for mice up to P14) -Depth limit established by autofluorescence -Limited number of channels (max. 2–3)	Dodt et al., 2007; Santi, 2011; Fiolka, 2019; Wang et al., 2019