er the past decade, confocal and multiphoton laser scanning microscopy have become standard for biomedical imaging on the cellular level. Because these techniques either suppress or don’t generate out-of-focus light, they produce clear images of the selected sample plane and a near-ideal signal-to-noise ratio for a given number of detected photons. Three-dimensional images of the sample can be obtained by recording a stack of images in subsequent focal planes. In recent years, scientists have used fluorescence lifetime imaging microscopy (FLIM) and multispectral detection with laser scanning microscopy. Multispectral detection can separate the fluorescence signals of various fluorescent species, and FLIM can separate the signals of a particular species in various states of interaction with the local environment. Fluorescence lifetime images of mouse kidney tissue were taken using two-photon excitation at 750 nm, in 16 wavelength channels from 350 to 537 nm. The lifetime from 1 to 2.4 ns is displayed by color. FLIM-based Förster resonance energy transfer (FRET) allows the resolution of interacting and noninteracting fractions of labeled proteins. The technique also can extract information from autofluorescence images and can provide quantitative results for protein-protein interactions. For a multispectral FLIM technique, it is important to avoid any time gating, time scanning or wavelength scanning. Gating, both in time and in wavelength, would result in losing most of the photons. This would require increasing the excitation power or acquisition time, which, in turn, would substantially increase photobleaching. Photobleaching not only changes the fluorescence lifetimes of the fluorophores, but also causes distortion in the scanned time or in the wavelength profiles. Moreover, multispectral FLIM should be able to resolve the components of the complex decay functions that are typical of autofluorescence and FRET applications. Multidimensional time-correlated single-photon counting solves these problems in an almost ideal way.1 The sample is excited by a laser that delivers picosecond or femtosecond pulses at a repetition rate of 10 to 100 MHz. The emission light is split into a spectrum by a polychromator and is projected on the photocathode of a multianode photomultiplier tube, which delivers an easily detectable pulse for each photon. The time-correlated single-photon counting module uses these pulses to construct a photon distribution over the laser pulse period. It also records the number of the photomultiplier tube channels (colors) that detected the photon and the coordinates of the scan. The result is an array of pixels, each of which contains a number of spectral channels. The spectral channels comprise a large number of time channels, each containing photon numbers for various times over the fluorescence decay period. Any scan rate The technique works at any scan rate, and the photon count rate does not have to be higher than the scan rate. The data acquisition runs over as many frames of the scan as necessary to obtain a satisfactory signal-to-noise ratio. The time-resolution is limited only by the transit-time spread in the photomultiplier tube. With the currently available multianode tubes, the width of the instrument-response function is about 150 ps (full width at half maximum). This is short enough to resolve fluorescence lifetimes down to 100 ps without noticeable loss in accuracy. Instrument-response function widths down to 30 ps can, in principle, be obtained with multichannel-plate photomultiplier tubes or arrays of single-photon avalanche photodiodes. Christoph Biskup at the Institute of Physiology II, Friedrich Schiller University, in Jena, Germany, took autofluorescence lifetime images of mouse kidney tissue in 16 wavelength intervals, from 350 to 537 nm (see figure). The color represents the fluorescence lifetime, and the brightness represents the number of photons per pixel. To keep the brightness within the printable range, all fluorescence images were normalized to 100 percent for the brightest pixel. The lifetime systematically decreases with increasing detection wavelength. The signal in channel 3 (around 375 nm) is dominated by second-harmonic generation in the tissue. Second-harmonic generation is an extremely fast process, as can be seen in the corresponding pixels displayed with a “decay time” shorter than 50 ps. The data was recorded by a multispectral FLIM system from Becker & Hickl of Berlin, connected to an LSM 510 NLO multiphoton microscope from Zeiss with an excitation wavelength of 750 nm. The acquisition time was 100 s and the count rate about 105 s—1. The FLIM system can process count rates as high as 5 x 106 s—1 and thus reduce the acquisition time to a few seconds. However, for the samples considered, the laser power that is required to obtain count rates higher than 105 s—1 causes unacceptable photobleaching and lifetime artifacts. Data analysis delivers lifetime images of single- and double-exponential decay parameters from data in selected wavelength intervals or single-wavelength channels. FLIM-FRET results can be obtained from the double-exponential lifetime analysis of a single donor image. Even in this conventional approach, multiwavelength FLIM data makes it easier to separate the donor fluorescence from acceptor fluorescence and autofluorescence. Meet the author Wolfgang Becker is president of Becker & Hickl GmbH in Berlin; e-mail: becker@becker-hickl.com. References 1. W. Becker (2005). Advanced Time-Correlated Single-Photon Counting Techniques. Springer Series in Chemical Physics, Vol. 81. Springer.