Error Analysis Of The Rapid Lifetime Determination Method For Double-Exponential Decays And New Windowing Schemes
by Kristin K. Sharman
- ABSTRACT: The rapid lifetime determination method (RLD) is a mathematical technique for extremely rapid evaluations of lifetimes in exponential decays. It has been applied in luminescence microscopy and single-molecule lifetime evaluation....
Anal. Chem.1999, 71,947-952
Dynamic Imaging Using Fluorescence Resonance Energy Transfer
by Masilamani Elangovan
- ABSTRACT: Recently, fluorescnce resonance energy transfer (FRET) and fluorescnce lifetime imaging microscopy (FLIM) have been the buzzwords for monitoring protein-protein interactions in living cells....
Biotechniques Vol. 32 No. 6 (2002)
Nanosecond Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy To Localize The Protein Interactions In A Single Living Cell
by Masilamani Elangovan
- ABSTRACT: Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescnce microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescent resonance energy transfer (FRET) to be used to study protein interactions in living spectrums....
Journal Of Microscopy, Vol. 205, Pt 1 January 2002, Pp. 3-14
Fluorescence Lifetime Imaging (FLIM) Of Green Fluorescent Fusion Proteins In Living Cells
by Ammasi Periasamy
- ABSTRACT: Fluorescence microscopy is an exception al tool for looling inside cells and tissues. Recent advances in fluorescence microscopy, including improved optics, sensitive fluorescent dyes, and high-sensitivity cameras, coupled with technological advances in computes and sophisticated software now permit quantitative measurement and noninvasive acquisition of spectroscopic information from a single living cell....
Methods In Molecular Biology, Vol. 183: Green Fluorescent Protein: Applications And Protocals
Time-Gated Fluorescence Microscopy For Clinical Imaging
by Assami Periasamy
- ABSTRACT: Fluorescence microscopy has been an essential tool for generations of biologists, allowing the examination of details of individual living or fixed cells. The development of a large number of fluorescnece dyes and the ability to attach them to specific molecular cell constituents allows real-time observation and quantification of the activities of these constituents in cells and tissues....
USA Microscopy And Analyses, March 1995 19
Time-Resolved Fluorescence Lifetime Imaging Microscopy Using A Picosecond Pulsed Tunable Dye Laser System
by Ammasi Periasamy
- ABSTRACT: The design and implementation of a time-resolved fluorescence lifetime imaging microscope (TRFLIM) for the biomedical sciences are described. The measurement of fluorescence lifetimes offers many benefits, among which is that they are independent of local signal intensity and concentration of the fluorophore and they provide visualization of the molecular environment in a single living cell.
Rev. Sci. Instrum. 67 (10), October 1996
Protein localization in living cells and tissues using FRET and FLIM
by Ye Chen, James D. Mills, & Ammasi Periasamy
- ABSTRACT: Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-.eld, confocal, or two-photon (2p) .uorescence resonance energy transfer (FRET) microscopy, ..........
Differentiation (2003) 71:528?41
Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy
by Yuansheng Sun, Richard N Day & Ammasi Periasamy
- ABSTRACT: Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from F?rster resonance energy transfer. We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT enhancer binding protein-a in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ~2 d.
Nature Protocol Vol. 6 No. 9, 2011.