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.
We report the creation of two-dimensional fluorescence lifetime images, based on a sinusoidally modulated image intensifier that is operated as a radio-frequency phase-sensitive camera, synchronized to a mode-locked and cavity dumped picosecond dye laser. By combining the image intensifier with a CCD camera and applying digital image processing, lifetime-selective signal suppression can be realized even for fluorophores with comparable lifetimes. This phase-sensitive technique can be used to create fluorescence lifetime images, that is, images in which the contrast is based upon the fluorescence lifetimes rather than upon local probe concentration and/or intensity. Because the lifetimes of many dyes are sensitive to the chemical environments surrounding the fluorophore, fluorescence lifetime imaging (FLIM) can reveal the local chemical composition and properties of the molecular environment that surrounds the fluorophore. As an example we created images of rhodamine 6G (4 ns) and rhodamine B (1.5 ns) solutions in which the difference in lifetimes results in 100% contrast, whereas the total fluorescence intensity is similar. In order to estimate the time resolution obtainable with our phase-sensitive imaging setup, we also performed measurements using distance-selective suppression of optical signals backscattered from laser-illuminated targets. In these studies, we have created distance-selective images with a resolution of 3.75 cm, which corresponds to a lifetime resolution of 0.25 ns for fast-decaying fluorophores. Since we observed nearly 100% contrast for this 0.25 ns difference, still smaller distances and/or lifetime differences could be observed, which seems to imply some advantages of phase fluorometric lifetime imaging. Using these same intensifiers in the pulse-gating mode one expects a typical 5 ns gating time, which result in a distance resolution of only 75 cm. The distance-selective imaging principle has the potential for manifold applications related to robotics, construction, and even to spacecraft maneuvering. Fluorescence lifetime imaging, in combination with fluorescence microscopy, can have numerous applications to biochemistry, biophysics, and cell physiology.
Measurements of the phase and modulation of amplitudemodulated light diffusely reflected by turbid media can be used to deduce absorption and scattering coefficients.
A flow cytometer capable of measuring fluorescence lifetimes by the phase shift method has been built and evaluated. Under optimal conditions, the resolution of the fluorescence lifetime measurement is shown to be under 200 picoseconds. Pulse intensity variations are normalized using limiting amplifiers and electronic filtering. Normalization of signal intensities provides a lifetime measurement that is independent of fluorescence intensity over at least a 50‐fold (17 dB) range in fluorescence intensity. The fluorescence lifetimes of unbound dye, fluorescent beads, cells stained with ethidium bromide, propidium iodide, and phycoerythrin‐conjugated monoclonal antibodies have been measured. The fluorescence lifetimes measured for these particles are well correlated with lifetime measurements made using a standard fluorimeter. Cells stained with ethidium bromide and propidium iodide at various nucleotide‐to‐dye ratios are shown to exhibit similar behavior to static cuvette measurements. The fluorescence lifetime parameter is also shown to resolve phycoerthyrin fluorescence from propidium iodide fluorescence. © 1993 Wiley‐Liss, Inc.
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