TABLE 5.

Comparison of microscopy techniques for cleared samples

Microscopy	Advantage	Disadvantage	Reference
Confocal microscopy	1. Achieving 3D imaging at high image contrast and resolution; 2. Enabling multispectral imaging without cross‐excitation; 3. Obtaining multicolor confocal images quickly.	1. Slow scanning speed; 2. The out‐of‐focus planes can also be illuminated during imaging, resulting in phototoxicity and sample photobleaching.	Conchello and Lichtman, 2005; Richardson and Lichtman, 2015
Two‐photon microscopy	1. Utilizing nonlinear techniques to generate signal contrast, reducing sample photobleaching; 2. Less sensitive to light scattering; 3. Adoption of near‐infrared light enhances tissue penetration and generates less phototoxicity and autofluorescence.	Utilization of laser scanning results in limited image acquisition rate.	Helmchen and Denk, 2005; Mertz, 2011; Lidke and Lidke, 2012
Light sheet microscopy	1. Allowing volumetric imaging at much higher speeds and signal‐to‐noise ratios; 2. Selective lower power illumination leads to less photobleaching and low phototoxicity.	Inability to image samples with sufficient pixel density at low magnification.	Keller and Ahrens, 2015; Hillman et al., 2019
SRS microscopy	1. Enabling fast super‐multiplex optical imaging without histological staining; 2. Utilization of nonlinear Raman effects improves the sensitivity and biocompatibility of spontaneous Raman imaging.	The penetration depth of SRS microscopy is limited in highly scattering tissues.	Wei et al., 2017; Wei et al., 2019