Table 2.

Fluorescence imaging techniques

Technique	Description	Benefits	Drawbacks
Colocalization	The observation of spatial overlap between different fluorescent labels, which reveals associations and interactions between two molecules [249,250]	• Can be conducted on widefield, confocal, and superresolution microscopes • Shows biomolecular associations and co-distributions	• Limited spatial and temporal resolution • Limited by resolution as the colocalization of two probes does not always signify association.
Fluorescence Recovery After Photobleaching (FRAP)	FRAP is used to determine the kinetics and diffusion of various biomolecules by intentionally photobleaching a portion of the sample and then observing how the fluorescence distribution returns to its previous state [71,251–254]	• Useful for finding ratios of bound and unbound molecules, as well as protein mobility • Turns photobleaching, which is generally avoided, into a desirable	• The photobleaching process can be destructive to the sample because of the high light intensity • Sometimes incomplete fluorescence recovery occurs due to obstruction of diffusion • A local temperature increase at the photobleached site can affect the calculated diffusion rate [255]
Fluorescence Correlation Spectroscopy (FCS)	FCS utilizes fluctuations in fluorescence intensity in small detection volumes in samples of low concentration to investigate molecular dynamics [186–194]	• Kinetics data can be measured in a living cell • Number of molecules of interest and their molecular brightness can be calculated	• Requires high labeling efficiency in order to get accurate kinetics data • Only counts the molecules in the observation volume, not the entire field of view
Single Particle Tracking (SPT)	SPT is a microscopy tool that allows the movement of individual particles to be followed within living cells. It provides information on molecular dynamics over time [256,257]	• Monitors the trajectories of individual biomolecules in living cells • Good for studying localization dynamics	• Requires extremely low fluorescent background and very bright labels • Requires highly sensitive cameras • Requires TIRF or HILO microscopes • Photobleaching (due to widefield imaging)
3D Orbital Tracking	3D Orbital Tracking uses an unique scanning pattern. Instead of exciting the molecule directly, the laser passing around the bright spot indirectly excites it, resulting in a longer imaging window [187,214]	• Minimal photobleaching • Can collect data for long periods of time	• Can only track one particle at a time • Only collects data on the molecule being tracked, not the rest of the field of view
Förster Resonance Energy Transfer (FRET)	FRET exploits the energy transfer that occurs between two chromophores that are in close proximity. The donor when in an excited state can transfer its energy to the acceptor through dipole-dipole coupling [258]. The excitation is accompanied by light emission and the transfer of energy is characterized by a loss of light emission. The efficiency of this transfer can be used to calculate small changes in distance between the chromophores [259].	• FRET is a nondestructive spectroscopic technique • Characterized molecular interactions with high accuracy (on the 1–10 nm scale)	• Low signal-to-noise ratio • Sensitivity of probes to pH, temperature, ionic concentration, etc.
Fluorescence Lifetime Imaging (FLIM)	FLIM specifically measures how long a fluorophore stays in an excited state before emitting a photon [260,261]	• Can detect molecular variations of fluorophores that are not apparent with spectral techniques alone • Ideal tool for removing background fluorescence intensity • Collects lifetime measurements for every pixel within the image	• Difficult to conduct in live cells because there are not enough photos per pixel • Requires in-depth data analysis