For FRAP analysis, a region of interest is defined and is bleached by brief illumination with high laser intensity. Once the region of interest is bleached, the progress of fluorescence recovery is recorded over time. Changes in intensity in the bleached region represent the sum of all movements of the fluorescent molecules, whether passive or active. The regeneration time (half-recovery period) is a measure of the speed of protein movement.
Fluorescence Loss in Photobleaching (FLIP)
For FLIP analysis, a region of interest is defined for bleaching. Brief illumination of the region with high laser intensity repeatedly during the course of imaging ensures it remains bleached. The progress of fluorescence decay in the adjacent non-bleached area is recorded. Changes in intensity in the non-bleached region represent the sum of all movements of the fluorescent molecules, whether passive or active. The decay of fluorescence (half-life) is a measure of the speed of protein movement.
Fluorescence Resonance Energy Transfer (FRET)
FRET analysis enables the quantitative temporal and spatial information about the binding and interaction between proteins, lipids, enzymes, DNA and RNA. FRET is a process in which energy is transferred from a fluorescent donor molecule to a fluorescent acceptor molecule. FRET can be detected as sensitized emission of the acceptor, where emission of an excited donor molecule specifically excites the acceptor molecule, thus increasing its emission. The efficiency of the transferred energy depends on the molecular distance between the two fluorophores. Energy transfer from donor to acceptor depletes or "quenches" the excited state population of the donor, and FRET will, therefore, reduce the fluorescence intensity of the donor. Photobleaching the acceptor to relieve this quenching of the donor (termed "acceptor photobleaching") offers another option for detecting FRET in vivo (Trinkle-Mulcahy et al., 2007).
Molecular or optical highlighters are fluorophores that can change color or emission intensity as a result of external photon stimulation or the passage of time. Examples of such fluorophores include:
Photo-activatable highlighters: produce no or little fluorescence, but dramatically increase their fluorescence intensity after activation by irradiation at a specific wavelength. For example, unconverted PA-GFP is optimally excited by 405 nm light, and demonstrates negligible absorbance by 488 nm light. After conversion with 405 nm light, PA-GFP absorption optimum is shifted to 504 nm, and will demonstrate an approximate 100-fold increase in green fluorescence by excitation with 488 nm light.
Photo-conversion highlighters: undergo a change in fluorescence emission profile upon irradiation at a specific wavelength. For example, the Dendra2 fluorophore emits light in the green range, but when 'converted' by irradiation in the UV range (ie 405 nm), will emit light in the red range.
Photo-switchable highlighters: have reversible on/off switching capabilities. For example, the Dronpa fluorophores can alternate between a dark state when irradiated with 405 nm light, and a fluorescent state when irradiated with 488 nm light.