Fluorescence Lifetime Imaging Microscopy (FLIM)
The fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon, which is an intrinsic property of a fluorophore. Fluorescence lifetime is sensitive to the local environment including pH, refractive index, temperature and insensitive to change in concentration and laser excitation intensity. There are different ways for measuring the fluorescence lifetime of a fluorophore: frequency domain (FD) and time domain (TD). Fluorescence lifetime measurements are performed both in single photon excitation and multiphoton excitation. Multiphoton microscopy provides several advantages in autofluorescence imaging over single photon confocal microcopy, such as less photobleaching, photodamage, scattering for live cell and tissue imaging. We have both one and two-three-photon lifetime imaging systems.
a. TD FLIM: The most common implementation of time domain (TD) fluorescence lifetime measurements are based on wide-field (gated camera), laser scanning microscopes both confocal and multi-photon mode with high speed detectors and electronics. Two- and Three-photon excitation lifetime imaging options are integrated with our existing Zeiss 780 multiphoton microscopy system. This is called time-correlated single photon counting (TCSPC) and the imaging board (SPC-150) and the FLIM hybrid detector (HPM-100-40) are from Becker & Hickl, Germany. We have two-channel FLIM detectors coupled to the Zeiss 780 NDD port. Both detectors allow simultaneous imaging of two fluorophores such as used for FRET or endogenous molecules such as tryptophan and NADH. Since the system optics transmits 300-1000 we are able to image auto-fluorescent tryptophan (300-400) with three-photon excitation (740nm).
b. Gated Camera: Ultrafast-gating image intensifier coupled to a CCD camera is used to acquire time-resolved images of proteins in living cells. This camera allows operating a gate width from 300 ps to 1 ms and a repetition rate from single shots to 110 MHz (PicoStar, Lavision, Germany). This gated image intensifier camera is synchronized with high-speed excitation laser pulses to trigger the camera gating pulse via a time-delay unit and synchronizing electronics. This gated camera can be coupled to any epi-fluorescence or TIRF microscopy system to implement FLIM or FLIM-FRET imaging. A rapid lifetime determination (RLD) method is used for the gating camera-based FLIM imaging. We developed data analysis software for single and double exponential decays.
c. FD FLIM: the fluorophores are excited with periodically intensity-modulated light to determine the modulation in the emission signal. The fluorescence emits at the same modulation frequency but phase shifts due to the delay caused by the lifetime of the fluorophore relative to the excitation time. This delay is measured as a phase shift (ϕ), and the modulation frequency (ω) in radian/sec. The lifetime of the fluorophore also causes demodulation to the modulated excitation by a factor mω, as a function of the decay time and light modulation frequency.
The ISS Alba FD FLIM, digital frequency-domain FastFLIM confocal (2 channels) system is equipped with a Nikon TE300 inverted epi-fluorescence microscope, Fianium SC-450 supercontinuum laser ranging from 450 to 2000 nm (usage wavelength is 450-900 nm), 375 nm laser line for NADH and NAD(P)H imaging and Two avalanche photodiode (APD) detectors.