Fluorescence Lifetime Imaging Microscopy (FLIM)
Fluorescence lifetime-resolved imaging microscopy (FLIM) refers to the technique of image formation in which the fluorescence lifetimes of emitting fluorophores are resolved for every image location in a specimen. By utilizing differences in excited-state lifetimes of the fluorophores as the contrast mechanism for image formation, many of the limitations of conventional fluorescence microscopy methods, based on spectrally resolved fluorescence intensity measurements, are avoided. The information represented in lifetime-resolved images may be the actual (or apparent) lifetimes, or another spectroscopic parameter that is able to be determined only by virtue of the fluorescence signal being lifetime-resolved.
Recommended Literature
- Bastiaens, P. I. H. and Squire, A. Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell. Trends in Cell Biology 9: 48-52 (1999).
- Levitt, J. A., Matthews, D. R., Ameer-Beg, S. M. and Suhling, K. Fluorescence lifetime and polarization-resolved imaging in cell biology. Current Opinion in Biotechnology 20: 28-36 (2009).
- Dong, C. Y., French, T., So, P. T. C., Buehler, C., Berland, K. M. and Gratton, E. Fluorescence-lifetime imaging techniques for microscopy. Methods in Cell Biology 72: 431-464 (2003).
- Krishnan, R. V., Masuda, A., Centonze, V. E. and Herman, B. Quantitative imaging of protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera. Journal of Biomedical Optics 8: 362-367 (2003).
- Suhling, K., French, P. M. W. and Phillips, D. Time-resolved fluorescence microscopy.Photochemical and Photobiological Sciences 4: 13-22 (2005).
- Duncan, R. R. Fluorescence lifetime imaging microscopy (FLIM) to quantify protein-protein interactions inside cells. Biochemical Society Transactions 34: 679-682 (2006).
- Squire, A., Verveer, P. J. and Bastiaens, P. I. H. Multiple frequency fluorescence lifetime imaging microscopy. Journal of Microscopy 197: 136-149 (2001).
- Chen, Y., Mills, J. D. and Periasamy, A. Protein localization in living cells and tissues using FRET and FLIM. Differentiation 71: 528-541 (2003).
- Festy, F., Ameer-Beg, S. M., Ng, T. and Suhling, K. Imaging proteins in vivo using fluorescence lifetime microscopy. Molecular BioSystems 3: 381-391 (2007).
- Yasuda, R. Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Current Opinion in Neurobiology 16: 551-561 (2006).
- Verveer, P. J., Squire, A. and Bastiaens, P. I. H. Global analysis of fluorescence lifetime imaging microscopy data. Biophysical Journal 78: 2127-2137 (2000).
- Breusegem, S. Y., Levi, M. and Barry, N. P. Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy. Nephron Experimental Nephrology 103: e41-e49 (2006).
Additional Literature Sources
- Becker, W., Bergmann, A., Haustein, E., Petrasek, Z., Schwille, P., Biskup, C., Kelbauskas, L., Benndorf, K., Klocker, N., Anhut, T., Riemann, I. and Konig, K. Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting. Microscopy Research and Technique 69: 186-195 (2006).
- Benninger, R. K. P., Hofmann, O., McGinty, J., Requejo-Isidro, J., Munro, I., Neil, M. A. A., deMello, A. J. and French, P. M. W. Time-resolved fluorescence imaging of solvent interactions in microfluidic devices. Optics Express 13: 6275-6285 (2005).
- Benninger, R. K. P., Koc, Y., Hofmann, O., Requejo-Isidro, J., Neil, M. A. A., French, P. M. W. and deMello, A. J. Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging. Analytical Chemistry 78: 2272-2278 (2006).
- Benninger, R. K. P., Hofmann, O., Onfelt, B., Munro, I., Dunsby, C., Davis, D. M., Neil, M. A. A., French, P. M. W. and deMello, A. J. Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems. Angewandte Chemie International Edition46: 2228-2231 (2007).
- Bird, D. K., Eliceiri, K. W., Fan, C. H. and White, J. G. Simultaneous two-photon spectral and lifetime fluorescence microscopy. Applied Optics 43: 5173-5182 (2004).
- Blum, C., Cesa, Y., Escalante, M. and Subramaniam, V. Multimode microscopy: Spectral and lifetime imaging. Journal of the Royal Society Interface 6: 535-543 (2009).
- Booth, M. J. and Wilson, T. Low-cost, frequency-domain, fluorescence lifetime confocal microscopy. Journal of Microscopy 214: 36-42 (2004).
- Calleja, V., Ameer-Beg, S. M., Vojnovic, B., Woscholski, R., Downwards, J. and Larijani, B. Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochemical Journal 372: 33-40 (2003).
- Clayton, A. H. A., Hanley, Q. S. and Verveer, P. J. Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy 213: 1-5 (2003).
- Clayton, A. H. A., Hanley, Q. S., Arndt-Jovin, D. J., Subramaniam, V. and Jovin, T. M. Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM).Biophysical Journal 83: 1631-1649 (2002).
- Connally, R. E. and Piper, J. A. Time-gated luminescence microscopy. Annals of The New York Academy of Sciences 1130: 106-116 (2008).
- Cremazy, F. G. E., Manders, E. M. M., Bastiaens, P. I. H., Kramer, G., Hager, G. L., van Munster, E. B., Verschure, P. J., Gadella, Jr., T. W. J. and van Driel, R. Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM. Experimental Cell Research 309: 390-396 (2005).
- Cross, A. J. and Fleming, G. R. Analysis of time-resolved fluorescence anisotropy decays.Biophysical Journal 46: 45-56 (1984).
- Dowling, K., Dayel, M. J., Lever, M. J., French, P. M. W., Hares, J. D. and Dymoke-Bradshaw, A. K. L. Fluorescence lifetime imaging with picosecond resolution for biomedical applications. Optics Letters 23: 810-812 (1998).
- Dowling, K., Hyde, S. C. W., Dainty, J. C., French, P. M. W. and Hares, J. D. 2-D fluorescence lifetime imaging using a time-gated image intensifier. Optics Communications 135: 27-31 (1997).
- Dunsby, C., Lanigan, P. M. P., McGinty, J., Elson, D. S., Requejo-Isidro, J., Munro, I., Galletly, N., McCann, F., Treanor, B., Onfelt, B., Davis, D. M., Neil, M. A. A. and French P. M. W. An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy. Journal of Physics D: Applied Physics 37:3296-3303 (2004).
- Elder, A. D., Frank, J. H., Swartling, J., Dai, X. and Kaminski, C. F. Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources. Journal of Microscopy 224: 166-180 (2006).
- Elson, D., Requejo-Isidro, J., Munro, I., Reavell, F., Suhling, K., Tadrous, P., Benninger, R., Lanigan, P., McGinty, J., Talbot, C., Treanor, B., Webb, S., Sandison, A., Wallace, A., Davis, D., Lever, J., Neil, M., Phillips, D., Stamp, G. and French, P. Time-domain fluorescence lifetime imaging applied to biological tissue. Photochemical and Photobiological Sciences 3: 795-801 (2004).
- Emptage, N. J. Fluorescent imaging in living systems. Current Opinion in Pharmacology 1:521-525 (2001).
- Esposito, A., Gerritsen, H. C. and Wouters, F. S. Fluorescence lifetime heterogeneity resolution in the frequency domain by lifetime moments analysis. Biophysical Journal 89:4286-4299 (2005).
- Gadella, Jr. T. W. J., Clegg, R. M. and Jovin, T. M. Fluorescence lifetime imaging microscopy: Pixel-by-pixel analysis of phase-modulation data. Bioimaging 2: 139-159 (1994).
- Grecco, H. E., Lidke, K. A., Heintzmann, R., Lidke, D. S., Spagnuolo, C., Martinez, O. E., Jares-Erijman, E. A. and Jovin, T. M. Ensemble and single particle photophysical properties (two-photon excitation, anisotropy, FRET, lifetime, spectral conversion) of commercial quantum dots in solution and in live cells. Microscopy Research and Technique 65: 169-179 (2005).
- Halmer, D., von Basum, G., Hering, P. and Murtz, M. Fast exponential fitting algorithm for real-time instrumental use. Review of Scientific Instruments 75: 2187-2191 (2004).
- Hanley, Q. S. and Clayton, A. H. A. AB - plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. Journal of Microscopy218: 62-67 (2005).
- Hanley, Q. S., Subramaniam, V., Arndt-Jovin, D. J. and Jovin, T. M. Fluorescence lifetime imaging: Multi-point calibration, minimum resolvable differences, and artifact suppression. Cytometry Part A 43: 248-260 (2001).
- Harpur, A. G., Wouters, F. S. and Bastiaens, P. I. H. Imaging FRET between spectrally similar GFP molecules in single cells. Nature Biotechnology 19: 167-169 (2001).
- Herman, P. and Vecer, J. Frequency domain fluorometry with pulsed light-emitting diodes.Annals of The New York Academy of Sciences 1130: 56-61 (2008).
- Jones, C. and Suhling, K. Mapping the refractive index sensing range of the GFP fluorescence decay with FLIM. Proceedings of SPIE 6098: 609807 (2006).
- Kremers, G., van Munster, E. B., Goedhart, J. and Gadella Jr., T. W. J. Quantitative lifetime unmixing of multiexponentially decaying fluorophores using single-frequency fluorescence lifetime imaging microscopy. Biophysical Journal 95: 378-389 (2008).
- Krishnan, R. V., Biener, E., Zhang, J., Heckel, R. and Herman, B. Probing subtle fluorescence dynamics in cellular proteins by streak camera based fluorescence lifetime imaging microscopy. Applied Physics Letters 83: 4658 (2003).
- Krishnan, R. V., Saitoh, H., Terada, H., Centonze, V. E. and Herman, B. Development of a multiphoton fluorescence lifetime imaging microscopy system using a streak camera.Review of Scientific Instruments 74: 2714-2721 (2003).
- Mizeret, J., Stepinac, T., Hansroul, M., Studzinski, A., van den Bergh, H. and Wagnieres, G. Instrumentation for real-time fluorescence lifetime imaging in endoscopy. Review of Scientific Instruments 70: 4689-4701 (1999).
- Ng, T., Squire, A., Hansra, G., Bornancin, F., Prevostel, C., Hanby, A., Harris, W., Barnes, D., Schmidt, S., Mellor, H., Bastiaens, P. I. H. and Parker, P. J. Imaging protein kinase C activation in cells. Science 283: 2085-2089 (1999).
- Niesner, R. and Gericke, K. Fluorescence lifetime imaging in biosciences: Technologies and applications. Frontiers of Physics in China 3: 88-104 (2008).
- Pelet, S., Previte, M. J. R. and So, P. T. C. Comparing the quantification of Förster resonance energy transfer measurement accuracies based on intensity, spectral, and lifetime imaging. Journal of Biomedical Optics 11: 034017 (2006).
- Pepperkok, R., Squire, A., Geley, S. and Bastiaens, P. L. H. Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Current Biology 9: 269-274 (1999).
- Periasamy, A., Elangovan, M., Elliot, E. and Brautigan, D. L. Fluorescence Lifetime Imaging (FLIM) of green fluorescent fusion proteins in living cells. Methods in Molecular Biology183: 89-100 (2002).
- Peter, M. and Ameer-Beg, S. M. Imaging molecular interactions by multiphoton FLIM.Biology of the Cell 96: 231-236 (2004).
- Peter, M., Ameer-Beg, S. M., Hughes, M. K. Y., Keppler, M. D., Prag, S., Marsh, M., Vojnovic, B. and Ng, T. Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophysical Journal 88: 1224-1237 (2005).
- Petty, H. R. Fluorescence microscopy: Established and emerging methods, experimental strategies, and applications in immunology. Microscopy Research and Technique 70: 687-709 (2007).
- Provenzano, P. P., Eliceiri, K. W. and Keely, P. J. Multiphoton microscopy and fluorescence lifetime imaging microscopy (FLIM) to monitor metastasis and the tumor microenvironment. Clinical and Experimental Metastasis 26: 357-370 (2009).
- Redford, G. I. and Clegg, R. M. Polar plot representation for frequency-domain analysis of fluorescence lifetimes. Journal of Fluorescence 15: 805-815 (2005).
- Requejo-Isidro, J., McGinty, J., Munro, I., Elson, D. S., Galletly, N. P., Lever, M. J., Neil, M. A. A., Stamp, G. W. H., French, P. M. W., Kellett, P. A., Hares, J. D. and Dymoke-Bradshaw, A. K. L. High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging. Optics Letters 29: 2249-2251 (2004).
- Schneider, P. C. and Clegg, R. M. Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications. Review of Scientific Instruments 68:4107-4119 (1997).
- Scully, A. D., MacRobert, A. J., Botchway, S., ONeill, P., Parker, A. W., Ostler, R. B. and Phillips, D. Development of a laser-based fluorescence microscope with subnanosecond time resolution. Journal of Fluorescence 6: 119-125 (1996).
- Seah, L. K., Wang, P., Murukeshan, V. M. and Chao, Z. X. Application of fluorescence lifetime imaging (FLIM) in latent finger mark detection. Forensic Science International 160: 109-114 (2006).
- Siegel, J., Elson, D. S., Webb, S. E. D., Parsons-Karavassilis, D., Leveque-Fort, S., Cole, M. J., Lever, M. J., French, P. M. W., Neil, M. A. A., Juskaitis, R., Sucharov, L. O. and Wilson, T. Whole-field five-dimensional fluorescence microscopy combining lifetime and spectral resolution with optical sectioning. Optics Letters 26: 1338-1340 (2001).
- Siegel, J., Suhling, K., Leveque-Fort, S. and Webb, S. E. D. Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore. Review of Scientific Instruments 74: 182-192 (2003).
- So, P. T. C., French, T., Yu, W. M., Berland, K. M., Dong, C. Y. and Gratton, E. Time-resolved fluorescence microscopy using two-photon excitation. Bioimaging 3: 49-63 (2001).
- Squire, A. and Bastiaens, O. I. H. Three dimensional image restoration in fluorescence lifetime imaging microscopy. Journal of Microscopy 193: 36-49 (1999).
- Strickler, S. J. and Berg, R. A. Relationship between absorption intensity and fluorescence lifetime of molecules. The Journal of Chemical Physics 37: 814 (1962).
- Striker, G., Subramaniam, V., Seidel, C. A. M. and Volkmer, A. Photochromicity and fluorescence lifetimes of green fluorescent protein. The Journal of Physical Chemistry B103: 8612-8617 (1999).
- Suhling, K., Siegel, J., Lanigan, P. M. P., Leveque-Fort, S., Webb, S. E. D., Phillips, D., Davis, D. M. and French, P. M. W. Time-resolved fluorescence anisotropy imaging applied to live cells. Optics Letters 29: 584-586 (2004).
- Suhling, K., Siegle, J., Phillips, D., French, P. M. W., Leveque-Fort, S., Webb, S. E. D. and Davis, D. M. Imaging the environment of green fluorescent protein. Biophysical Journal 83: 3589-3595 (2002).
- Tadrous, P. J. Methods for imaging the structure and function of living tissues and cells: 2. Fluorescence lifetime imaging. The Journal of Pathology 191: 229-234 (2000).
- Tramier, M., Kemnitz, K., Durieux, C. and Coppey-Moisan, M. Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein. Journal of Microscopy 213: 110-118 (2004).
- Tramier, M., Zahid, M., Mevel, J., Masse, M. and Coppey-Moisan, M. Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microscopy Research and Technique 69: 933-939 (2006).
- van Geest, L. K. and Stoop, K. W. J. FLIM on a wide field fluorescence microscope.International Journal of Peptide Research and Therapeutics 10: 501-510 (2003).
- Verveer, P. J. and Bastiaens, P. I. H. Evaluation of global analysis algorithms for single frequency fluorescence lifetime imaging microscopy data. Journal of Microscopy 209: 1-7 (2003).
- Verveer, P. J., Squire, A. and Bastiaens, P. I. H. Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data. Journal of Microscopy 202: 451-456 (2001).
- Wang, Y., Shyy, J. Y. J. and Chien, S. Fluorescence proteins, live-cell imaging, and mechanobiology: Seeing is believing. Annual Review of Biomedical Engineering 10: 1-38 (2008).
- Webb, S. E. D., Gu, Y., Leveque-Fort, S., Cole, S. M. J., Dowling, K., Jones, R., French, P. M. W., Neil, M. A. A., Juskaitis, R., Sucharov, L. O. D., Wilson, T. and Lever, M. J. A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning. Review of Scientific Instruments 73: 1898-1907 (2002).
- Wouters, F. S. and Bastiaens, P. I. H. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Current Biology 9: 1127-1130 (1999).