Table 1.

Possibilities and technical characteristics of the optical methods used in vivo/ex vivo to determine skin penetration.

Optical Method		Imaging Method (2D/3D)	Screening Depth, µm	Lateral/Axial Resolution	Penetration of Non-Particulate Substances	Penetration of Particulate Substances	Possibility to Quantify the Substances Used
FCLSM	in vivo	Yes	≈100 µm	<1 µm/ <5 µm	Yes 1,2 (low sensitivity)	Yes 1,2 (low sensitivity)	Yes [73]
	ex vivo	Yes
RCLSM	in vivo	Yes	≈150 µm		No	Yes (low sensitivity)	No
	ex vivo	Yes
2PT-AF	in vivo	Yes	≈150 µm	<1 µm/ <2 µm	Yes 2 (low sensitivity)	Yes 2 (low sensitivity)	No
	ex vivo	Yes
2PT-FLIM	in vivo	Yes			Yes 3 (high sensitivity)	Yes 3 (high sensitivity)
	ex vivo	Yes
SHG	in vivo	Yes			No	Yes (high sensitivity)
	ex vivo	Yes
2PT-CARS	in vivo	Yes			Yes (low sensitivity) 4	Yes (low sensitivity) 4
	ex vivo	Yes
3PT	in vivo	Yes	<900 µm	<1 µm/ <2 µm	No	No	No
	ex vivo	Yes
CRM	in vivo	No 5	≈50 µm	<5 µm	Yes (high sensitivity)	Yes (high sensitivity)	Yes [207,209]
	ex vivo	Yes
SERS	in vivo	No 5	≈50 µm	<5 µm	No 6	Yes 7 (high sensitivity)	No
	ex vivo	Yes
SRS	in vivo	Yes	≈150 µm	<2 µm	Yes (high sensitivity) 4	Yes (high sensitivity) 4	Yes [160]
	ex vivo	Yes
OCT	in vivo	Yes	<2 mm	<2 µm/ <5 µm	No	Yes (low sensitivity) 8	No
	ex vivo	Yes

¹ The applied substances should be inherently fluorescent or covalently bound with a fluorescent dye; ² The applied substances should have intense AF whose intensity exceeds skin AF; ³ The applied substances should have AF lifetimes that are different from the AF lifetimes of the skin; ⁴ Sensitivity will be higher for substances whose Raman spectrum do not overlap with the skin spectrum; ⁵ Only 1D (z-stack point measurements) imaging is possible; ⁶ Non-particulate substances can be identified with SERS-labeled detection microneedles or with TERS (low-invasively on tape strips); ⁷ Only the detection of SERS-active nanostructures is possible; ⁸ The higher the density of target substances, the higher the contrast of OCT images.