Table 1.

Summary of the techniques presented, highlighting their advantages and disadvantages for fate studies in vitro and in vivo.

Techniques	Advantages	Disadvantages	Experimental Type	References
FC	Quantitative measurements of NC/drug uptake in a cell population measuring fluorescence at single cell level.	Requires fluorescence labelling. Cannot discriminate easily between NPs. inside or on the surface of cells.	In vitro	[34,35,36,37,47]
CLSM	Able to visualize the location of NCs and drugs inside cell organelles.	Requires fluorescence labelling. Can give false results due to detached dyes from NCs.	Mainly in vitro	[23,24,25,26,27]
FRET	Can determine the proximity of two fluorescence molecules in vitro and in vivo. can be used for studying release of drugs or degradation of NPs	Requires labelling and sometimes complex. Molecular design.	In vitro/in vivo	[50,51]
FCS/FCCS	Allows for studying diffusion of fluorescent molecules and the temporally correlate the association of labelled species. Can be applied for studying degradation of NCs or Drug release.	Requires fluorescence labelling. Photobleaching can difficult measurements	In vitro	[63,64,79,80]
FLIM	Imaging is based on measuring lifetime of fluorescence molecules that can be traced even with low intensity. Lifetime measurements are sensitive to environmental conditions such as pH, ionic strength, temperature.	Requires fluorescence labelling. Can give false results due to detached dyes from NCs.	In vitro/in vivo	[81,82,83,84,85]
Fluorescence Imaging	Gives precise information about NCs/drug distribution biodistribution (in vivo).	Long exposure to fluorescent light can cause bleaching. Dye detachment can lead to a false localization of NPs or drugs. Not quantitative.	In vivo	[106,107,108]
Raman	Minimal sample preparation. Non-invasive. Non-destructive. Label-free manner visualization of NPs and drug in cells and tissues. Co localization studies of NPs and drug without additional labelling.	Limited confocality. Time consuming. Fluorescence can interfere with measurements.	In vitro	[94,96]
PET/SPECT	Based on the detection of radioactive element. Quantitative. In vivo biodistribution of NPs/drugs can be quantitatively determined.	Requires radiolabelling nanomaterials. Can give false. Information if radioisotopes detach. Molecules and imaging techniques are not easily accessible.	In vivo	[118,119,120,121,122,123]