FRAP and Photoactivation

Fluorescence recovery after photobleaching (FRAP) is an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane diffusion and protein binding. In addition, surface deposition of a fluorescing phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy. FRAP-based techniques have been developed to investigate the 3-dimensional diffusion and binding of molecules inside the cell. FRAP can be used to determine if a protein is able to move within a membrane (high percent recovery with a fast mobility), or whether it is tethered other structural components of the cell (low percent recovery with a slow mobility).

By using two-photon excitation FRAP someone can study the three-dimensional mobility of fluorescent molecules with three-dimensional resolution at a micrometer scale. As unbleached fluorescent molecules diffuse in, the fluorescence is monitored with a lower laser power to measure fluorescence recovery. From the time course of fluorescence recovery, the diffusion coefficient of the fluorescent molecule can be measured.

Using 2PE photobleaching, diffusion of biochemical substances through the spine neck has been measured^1,2,3.

Svoboda et al.⁴ used this technique to study the diffusional exchange between dendritic spines and shafts of CA1 neurons in rat hippocampal slices. A somehow opposite technique to FRAP, photoactivation of caged fluorophores^4,5 and photo-activatable genetically encoded fluorescence proteins⁶, can also be used to measure the diffusion and trafficking of tagged molecules⁷. Photoactivation may offer advantages over FRAP. First of all, because most photo-activatable fluorescent proteins produce little fluorescence before photoactivation, the measurements have intrinsically higher signal-to-noise ratios⁸. Secondly, photoactivation may produce fewer free radicals and thus induce less phototoxicity⁸.

References:

1. Majewska, A., Tashiro, A., and Yuste, R. (2000a). Regulation of spine calcium dynamics by rapid spine motility. J. Neurosci. 20, 8262– 8268.
2. Pologruto, T.A., Yasuda, R., and Svoboda, K. (2004). Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J. Neurosci. 24, 9572–9579.
3. Sobczyk, A., Scheuss, V., and Svoboda, K. (2005). NMDA receptor subunit-dependent [Ca2+] signaling in individual hippocampal dendritic spines. J. Neurosci. 25, 6037–6046.
4. Svoboda, K., Tank, D.W., and Denk, W. (1996). Direct measurement of coupling between dendritic spines and shafts. Science 272, 716– 719.
5. Mitchison, T.J., Sawin, K.E., Theriot, J.A., Gee, K., and Mallavarapu, A. (1998). Caged fluorescent probes. Methods Enzymol. 291, 63–78.
6. Lukyanov, K.A., Chudakov, D.M., Lukyanov, S., and Verkhusha, V.V. (2005). Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell. Biol. 6, 885–891.
7. Bloodgood, B.L., and Sabatini, B.L. (2005). Neuronal activity regulates diffusion across the neck of dendritic spines. Science 310, 866–869.
8. Karel Svoboda and Ryohei Yasuda, Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience, Neuron 50, 823–839, June 15, 2006 2006 Elsevier Inc.