Fluorescence lifetime imaging

Abstract

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.

A Method for Chorioretinal Oxygen Tension Measurement

“…A frequency-domain approach was used for the measurement of phosphorescence lifetime as previously described. 13,[25][26][27] In this approach, the light that is used for excitation of the probe and the sensitivity of the camera that is used for detection of the phosphorescence emission are independently modulated. By varying the phase relationship between the two modulators, phosphorescence phase and lifetime are derived.…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…fluorescence lifetime imaging -FLIM, allow a complete, bioscientifically relevant picture of the samples under study 6,8,26,28 .…”

“…However, this emission does not precisely follow the excitation but rather shows phase delays and amplitude changes which are determined by the fluorescence decay law of the sample, i.e. the phase of the emission is shifted to later time points as compared to the excitation light, whereas the peak-to-peak height of the modulated emission is decreased relative to that of the modulated excitation [6][7][8]26,28 .…”

“…As the modulation frequency is increased, the finite lifetime of the excited state prevents the emission from precisely following the excitation. Thus, for a single-exponential decay, the phase shift and the demodulation factor measured at a particular frequency are related to the fluorescence lifetime as follows 6,7,28 :…”

“…quenching or FRET, but it is hardly affected by fluctuations of the chromophore concentration, which implies highaccuracy calibration-free measurements. Thus, fluorescence lifetime imaging (FLIM) can in many cases counteract the disadvantages of steady-state techniques 6,7 and, consequently, has become a versatile tool in the quantitative measurement of cellular parameters as well as in monitoring and quantifying molecular phenomena within the cell.…”