Lifetime-selective fluorescence imaging using an rf phase-sensitive camera

Abstract

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.

Indicators for optical oxygen sensors

“…One of the first works on intensity-based phosphorescence imaging was published in 1988 and used to measure oxygen distribution in perfused tissue [192]. Lifetime imaging techniques were developed for the first time two decades ago, and both timedomain (pulsed) [229] and frequency-domain (phase-resolved) [125] have been described; in 1997, the first noninvasive technique to measure oxygen concentration on nonplanar surfaces (e.g., human skin) was described [92].…”

A wide-field TCSPC FLIM system based on an MCP PMT with a delay-line anode

“…Fluorescence Lifetime Imaging (FLIM) techniques can be divided into time-domain and frequency-domain techniques, 6-9 photon counting 10,11 and analog techniques, 6,[12][13][14][15] and point-scanning 7,16 and widefield imaging techniques. 6,12,15,17 It also matters whether a technique acquires the signal waveform in a few time gates 10 or in a large number of time channels 11,18 and whether this happens simultaneously 10,11,18 or sequentially. 12,15,17 Virtually all combinations are in use.…”

Detectors for single-molecule fluorescence imaging and spectroscopy

“…FLIM can be performed in two different ways: using time-domain measurement, or frequencydomain measurements (43,44,51). The time-domain approach can be implemented using a time-correlated single-photon counting (TCSPC) confocal setup, which requires rasterscanning the sample to build an image.…”