Special cases of fluorescence#
Go-to source for information on all related to fluorescence is “Principles of fluorescence spectroscopy” by Lakowicz. Here we briefly review some of the materials covered in that book.
Split-proteins#
Splitting fluorescent protein (expression as two distinct parts) allows study of interaction between proteins or other molecular dynamics within the cell. In such experiments, parts of e.g. GFP are expressed independently. Normally, these parts are not reconstitute into a whole fluorescent protein, thus providing no signal readout.
However, if the ends of parts are connected to proteins that interact, this interaction brings split GFP fragments close enough for them to reconstitute functional fluorescent protein.
Molecular Sensors#
In similar fashion, starting with non-functional fluorescent protein that becomes functional under specific molecular conditions lead to development of molecular sensors based on fluorescent proteins. One such example is calcium indicator GCaMP.
The GFP protein is circularly permuted (cpGFP) and its ends are fused to calcium-binding protein domain of calmodulin (CaM). Unless calcium binds to CaM domain, the cpGFP assumes non-fluorescent molecular conformation. When concentration of calcium ions gets higher such as during action potential firing in neurons or egg activation more GCaMP molecules become fluorescent thus increasing detected signal.
Resonance energy transfer (FRET)#
Fluorescence recovery after photobleaching (FRAP)#
Fluorescent molecules can relax from excited state through several mechanisms. Some of these mechanisms such as fluorescence allow for the molecule to return to “default” ground state to be excited again. Other possible states, such as triplet allow for chemical reaction to occur that permanently renders molecule non-fluorescent thus causing “bleaching”.
FRAP experiments allow tracking of molecular movement in part of tissue by first purposefully bleaching most of the present molecules and then following increase (recovery) of signal due to mobile molecules coming in.
Blinking#
Another case of non-fluorescent state causes blinking - that is recoverable loss of fluorescence. Blinking is caused by temporary molecular transition to non-fluorescent state followed by recovery to ground state.
Recovery can happen on it’s own (like in STORM, stochastic optical reconstruction microscopy) or caused by external light stimuli (PALM, photoactivated localization microscopy).