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.

FIG. 1.

Intuitive presentation of the concept of fluorescence lifetime imaging (FLIM). The object is assumed to have two regions that display the same fluorescence intensity (I₁ = I₂) but different decay times, τ₂ > τ₁. (a) Object; (b) steady-state image; (c) gray-scale or color lifetime image; (d) lifetime surface.

FIG. 2.

Schematic diagram of a FLIM experiment. The “object” consists of a row of four cuvettes and has regions with different decay times, τ₁ to τ₄. This object is illuminated with intensity-modulated light. The spatially varying emission is detected with a phase-sensitive image intensifier, which is imaged onto a CCD camera. The laser system is a cavity-dumped dye laser, which is synchronously pumped by a mode-locked and frequency-doubled NdYAG laser.

FIG. 3.

Emission spectra of standard fluorophores used for FLIM. The emission was observed through a Corning 3-75 filter. Excitation was at 355 nm.

FIG. 4.

Phase-sensitive images of standard fluorophores at 49.53 MHz.

FIG. 5.

Phase-sensitive intensities of standard fluorophores at various detector phase angles.

FIG. 6.

Phase angle (top) and modulation (bottom) of the standards observed at 49.53 MHz.

FIG. 7.

Phase and modulation lifetime images of the standards observed at 49.53 MHz.

FIG. 8.

Phase angle (top) and modulation images (bottom) of the standards observed at 34.28 MHz (left) and 64.77 (right).

FIG. 9.

Phase-sensitive images of acrylamide-quenched Y_t-base for various detector phase angles.

FIG. 10.

Phase-sensitive intensities of Y_t-base in the presence of 0.4 (○), 0.2 (Δ), 0.1 (●), and 0.0 m acrylamide (▲).

FIG. 11.

Phase and modulation images of Y_t-base with acrylamide quenching.

FIG. 12.

Phase and modulation lifetimes images of Y_t-base with various concentrations of acrylamide.

FIG. 13.

Intuitive description of phase suppression. In a difference image with ΔI(θ_D + 180°) – I(θ_D), a component with θ = θ_D is completely suppressed. Components with longer lifetimes (phase angles) appear as negative spots, and those with shorter lifetimes (phase angles) appear to be positive.

FIG. 14.

Phase-suppression images of Y_t-base quenched by acrylamide. In the phase-sensitive image (a) the detector phase was 203.2°. The difference images were calculated using (b) I(48.1°) – I(338.9°), τ_s = 3.53 ns and (c) I(268.3°) – I(48.1°), τ_s = 0.94 ns.

FIG. 15.

Gray-scale suppression images of acrylamide-quenched Y_t-base. (a) Phase-sensitive image with ${\theta }_{\mathrm{D}}^{\prime }={203.3}^{{}_{{}^{°}}}$. (b) Difference image with τ_s = 0.9 ns using I(268.3°) – I(48.1°). (c) Difference image with τ_s = 3.5 ns using I(48.1°) – I(338.9°).