Fluorescence microscopy single-photon timing

There has been a considerable decline in the number of papers which deal with the details of techniques of measurement of fluorescence decay. This is no doubt due to the fact that the alternative methods are now essentially well established. Nevertheless a microcomputerized ultrahigh speed transient digitizer and luminescence lifeline instrument has been described . A very useful multiplexed array fluorometer allows simultaneous fluorescence decay at different emission wavelength using single photon timing array detection . Data collection rates could approach that for a repetitive laser pulse system and the technique could be usefully applied to HPLC or microscopy. The power of this equipment has been exemplified by studies on aminotetraphenylporphyrins at emission wavelengths up to 680 nm. The use and performance of the delta function convolution method for the estimation of fluorescence decay parameters has been... 

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

Duncan, R.R., Bergmann, A., Cousin, M.A., Apps, D.K., and Shipston, M.J. et al. 2004. Multidimensional time correllated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (Aim) to Detect Fret in Cells. J. Microsc. 215 1. 

Figure 11.21 StimuLated emission depletion (STED) microscopy. The sample is excited using single-photon excitation (PUMP pulse) in a confocal microscope arrangement. A time-delayed DUMP pulse selectively depletes close to 100% of the exdted state population in a region around the focus of the PUMP pulse. Using this approach. Hell and co-workers were able to obtain a 5-fold reduction in the fluorescent spot size in the vertical (Z-direction) and a greater than a 2-fold reduction in the horizontal Y/X) direction, leading to a final image size of 97 by 104 nm...

Figure 25. Fluorescence microscopy of a single molecule of terry-lene in hexadecane. The emission from a round sample of a diameter of 100 pm is shown with a fixed laser excitation wavelength of 572.379 nm with intensity 20 mW/ cm- and accumulation time 10 s. The integrated intensity under the peak is 4.700 photons per second and molecule and each pixel is 0.7 X 0.5 pm [47].

Keywords Autofluorescence Decay time Delayed fluorescence Fluorescence lifetime imaging microscopy Lanthanides Phosphorescence Time-correlated single photon counting Time-resolved fluorescence microscopy... 

Keywords Fluorescence lifetime imaging microscopy Frequency domain Time-correlated single photon counting Fluorescence resonance energy transfer Protein-protein interactions... 

Cr=crystal Sm=smectic CrSmB = crystal smectic B N=nematic Ch=cholesteric I=isotropic fluorescence = steady state fluorescence SPC = time-resolved single photon counting CPF=circularly polarized fluorescence UV-vis = UV-visible absorption spectrophotometry DSC=differential scanning calorimetry OM = optical microscopy XRD = X-ray diffraction EPR=electron paramagnetic resonance NMR=nuclear magnetic resonance. 

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