FLIM Publications

Error Analysis Of The Rapid Lifetime Determination Method For Double-Exponential Decays And New Windowing Schemes 

  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 
PDF  


Dynamic Imaging Using Fluorescence Resonance Energy Transfer 
  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) 
PDF 


Nanosecond Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy To Localize The Protein Interactions In A Single Living Cell 
  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 
PDF  


Fluorescence Lifetime Imaging (FLIM) Of Green Fluorescent Fusion Proteins In Living Cells 
  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 
PDF  


Time-Gated Fluorescence Microscopy For Clinical Imaging 
  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 
PDF  


Time-Resolved Fluorescence Lifetime Imaging Microscopy Using A Picosecond Pulsed Tunable Dye Laser System 
  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 
PDF  


Protein localization in living cells and tissues using FRET and FLIM 
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 
PDF 


Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy 
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. 
PDF