Two-photon fluorescence lifetime imaging

Hanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM (2002) Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient. BiophysJ 83 1682-1690. 

As distinct from sohd supports such as gold or silver, mercury imparts lateral mobihty to hpid monolayers directly self-assembled on its surface, because of its liquid state. This is demonstrated by rapid spontaneous phase separation, with microdomain formation, in a hpid mixture monolayer self-assembled on top of a DPTL thiolipid monolayer tethered to a mercury microelectrode [30]. The presence of microdomains was directly verified from the images of the distal hpid monolayer obtained using two-photon fluorescence lifetime imaging microscopy. 

Fig. 5.78 Two-photon autofluorescence lifetime image of aortic tissue. Top Intensity image, lifetime image of average lifetime, lifetime distribution. Bottom Fluorescence decay in indicated pixel and double exponential fit with 81% of 294 ns and 18.7% of 2.26 ns. From [39]...

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. and Barry, N. (2003). Fluorescence lifetime imaging for the two-photon microscope time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381-90. 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

Sytsma, J., Vroom, J.M., de Grauw, C.J., and Gerritsen, H.C. 1998. Time-gated fluorescence lifetime imaging using two photon excitation. J. Microsc. 191 39-51. 

Fig. 5.82 Fluorescence lifetime images of a transverse section though the medulla of a Cynomolgus monkey kidney acquired at the (a) 480-, (h) 510-, (c) 550-, and (d) 580-nm wavelength components of the emission spectrum. The sample was stained with methyl green and imaged by two-photon excitation at a wavelength of 920 nm. From [60], images courtesy of Damian Bird, University of Wisconsin...

Y. Chen, A. Periasamy, Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization, Microsc. Res. Tech. 63, 72-80 (2004)... 

H.C. Gerritsen, K. de Grauw, One- and Two-Photon confocal fluorescence lifetime imaging and its application, in A. Periasamy (ed.) Methods in Cellular Imaging, Oxford University Press, 309-323 (2001)... 

Wilms, C. D., Schmidt, H. and Eilers, J. (2006). Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis. Cell Calcium 40, 73-9. 

A typical image obtained by nondescanned detection and two-photon excitation is shown in Fig. 5.78. The autofluorescence of aortic tissue was excited at 800 nm. The figure shows the intensity image, an image of the average lifetime, and the lifetime distribution over the pixels. The fluorescence decay displayed for a selected pixel is multiexponential, as is typical for autofluorescence. 

A potential application of multimodule systems is high-speed two-photon multibeam scaiming systems [53, 77]. FLIM systems with 4, 8 or even 16 beams and the same number of parallel TCSPC channels appear feasible. The problem is to direct the fluorescence signals from the individual beams to separate PMTs or separate charmels of a multianode PMT. If this problem is solved, two-photon lifetime images can be recorded with unprecedented speed and resolution. 

TCSPC with two-dimensional position-sensitive detection can be used to acquire time-resolved images with wide-field illumination. The complete sample is illuminated by the laser and a fluorescence image of the sample is projected on the detector. For each photon, the coordinates in the image area and the time in the laser pulse sequence are determined. These values are used to build up the photon distribution over the image coordinates and the time (see Fig. 3.12, page 40). The technique dates back to the 70s [312] and is described in detail in [262]. Lifetime imaging with a TCSPC wide-field system and its application to GFP-DsRed FRET is described in [162]. A spatially one-dimensional lifetime system based on a delay-line MCP is described in [509]. 

A considerable improvement can be expected from the application of TCSPC to autofluorescence imaging of tissue. As mentioned in the application section of this book, the fluorescence lifetime helps not only to separate the different types of fluorescence but also to characterise the state of their binding to proteins, lipids, or DNA, as well as the oxygen saturation or the pH of the tissue. The first steps have been taken in time-resolved two-photon microscopy. The same basie optieal principles are leading to imaging of macroscopic samples. 

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