Flourescence-Lifetime+Imaging+Microscopy+(FLIM)

By Jessica Spencer

 Fluorescence Lifetime Imaging Microscopy (FLIM) is a generally new microscopy technology that enables researchers to incorporate five to six colored dyes simultaneously using the rate at which the fluorescence decays. The name comes from the fact that in typical fluorescent imaging, the intensity is used, but in FLIM the lifetime of the fluorophore signal is used. This is opposed to the conventional use of one color, and FLIM is also better for picturing what is around the molecule as well, not just the molecule itself [1]. The technique is An example of the difference between FLIM and regular confocal imaging can be seen below in Figure 1 [2]. The purpose of this technique is that it enables you to use various fluorescent colorations instead of just one when imaging, and the technique came about in the mid 1990s. Two types of FLIM are Time Domain and Frequency Domain. Figure 1.  //Frequency Domain and Time Domain FLIM //  Frequency Domain FLIM is when the sample that is being used for imaging is excited with intensity modulated light, and the emission of the fluorophore is dependent on the frequency of the excitation. Time Domain FLIM goes hand in hand with Time-correlated single-photon counting (TCSPC). TCSPC is used to distinguish individual photons, and is used to record the intensity based on time for excitation of a flash of light [6].

//Recent Research //  The first article that incorporates this technique is “Ato protein interactions..” [5]. The important fact that the FLIM revealed in this article was that it allowed the researchers to follow interactions between Ato proteins that were among distinctive areas of the yeast cells. This is also the importance of the technique in this article in that without the ability of FLIM to label individual portions of specific cells, the researchers would only be able to focus on the whole rather than the individual parts that they were interested in.  The second article that incorporates this technique is “Detection of lipid…” [4]. The importance of the technique in this article was that for their experimentation they needed to be able to see more than just surface in terms of the lipids that they were researching, which FLIM enables them to see various areas, not just flat structures. The important fact that FLIM revealed in this article was that in terms of studying the complex compartments and domains of lipids, FLIM is a very successful technique.  The third article that incorporates this technique is “Non-invasive imaging…” [3]. The importance of the technique in this article was that it allowed the researchers to specify and detect chemicals and nanoparticles that were applied to the skin. The technique allowed researchers to measure penetrance of compounds in skin. **References: ** [1] Olympus Corporation. (2004-2009). Flourescence lifetime imaging microscopy (FLIM), //Applications In Confocal Microscopy //, retrieved March 21, 2013 from http://www.olympusfluoview.com/applications/flimintro.html [2] Pudavar, H. (2009). Fluorescence lifetime imaging (FLIM). //Leica Microsystems Inc.// [3] Roberts, M.S., Dancik, Y., Prow, T.W., Thorling, C.A., Lin, L.L., Grice, J.E., Robertson, T.A., Konig, K., Becker, W. (2011). Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy. //<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">European Journal of Pharmaceutics and //// <span style="font-family: 'Times New Roman',serif; font-size: 12pt;"> Biopharmaceutics //<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">, 469-488. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">[4] Stockl, M.T., Herrmann, A. (2010). Detection of lipid domains in model and cell membranes <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">by fluorescence lifetime imaging microscopy. //<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">Biochimica et Biophysica Acta //<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">, 1444-1456. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">[5] Strachotova, D., Holoubek, A., Kucerova, H., Benda, A., Humpolickova, J., Vachova, L., <span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;"> Palkova, Z. (2012). Ato protein interactions in yeast plasma membrane revealed by fluorescence lifetime imaging (FLIM). //<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">Biochimica et Biophysica Acta //<span style="font-family: 'Times New Roman',serif; font-size: 12pt; line-height: 1.5;">, 2126-2134. <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">[6] Wahl, M. (2009). Time-correlated single photon counting. //PicoQuant.// P1-10.