TABLE 1.

Imaging techniques (Mempel et al., 2003; Masedunskas et al., 2012; Weigert et al., 2013; Marques et al., 2015b; Vielreicher et al., 2017).

Excitation	Technique	Microscopy	Light source	Detection	Advantages	Disadvantages
Single-photon	Widefield		Mercury lamp/LED	CCD	• Fast acquisition • Low cost	• Limited depth • Phototoxicity
		RLOT	Xenon lamp	CCD	• Detection of fast dynamic activity in the absence of specific fluorophores	• Limited to translucent tissue
	Confocal	LCSM	Lasers	PMT	• High spatial resolution • 3D sectioning	• Limited depth • Slow acquisition • Phototoxicity • Relatively high cost and smaller field of view
		SDCM	Lasers	CCD	• Fast acquisition • Low phototoxicity • 3D sectioning	• Limited depth • Faster acquisition • Pinhole crosstalk reduces the resolution
Two or more photon	Multiphoton	Two-photon	Lasers	PMT	• Extended depth • No off-focus emissions	• High cost • Slow acquisition
		Three-photon	Lasers	PMT	• Deep tissue imaging • Improved signal to background ratio	• High cost • Slow acquisition
		SHG and THG	Lasers	PMT	• No energy absorption • Label-free imaging of collagen, myosins, myelin, and lipids

LED, light-emitting diode; RLOT, reflected light oblique transillumination; CCD, charge-coupled device; LCSM, laser confocal scanning microscopy; PMT, photomultiplier tube; SDCM, spinning disk confocal microscopy; SHG, second-harmonic generation; THG, third-harmonic generation.