One multiphoton microscope is good, but more can be better, especially when it comes to speed. A team of researchers has combined multifocal, multiphoton excitation with time-correlated single-photon-counting detection to increase the image acquisition rates to 16 times that of single-beam systems. With this, the investigators have a tool that they hope to use to study the protein-to-protein interactions that occur when immune cells examine target cells. The scientists represent Imperial College London, Cancer Research UK of London and GlaxoSmithKline of Harlow, UK.This schematic illustrates an experimental multiphoton, multifocal microscope setup. Output from a Ti:sapphire laser travels through the beamsplitter and the scanner to produce 16 excitation spots that are scanned across a sample. The resulting fluorescence is detected with a 16-element photomultiplier array. P = pinhole; Pol = rotatable polarizing beamsplitter; L = lens; MUX = beam multiplexer; λ = half-wave plate; IP = image plane; PR = prism. Images reprinted with permission of Optics Express.A better understanding of those interactions would help biological research and the development of therapeutics. “We hope that this microscope will provide faster imaging of these intracellular events,” said Chris Dunsby, a member of the college’s photonics group.One way to study such events is to use Förster resonance energy transfer (FRET), in part because it provides information on molecular interactions. FRET signals can be read via fluorescence lifetime imaging microscopy (FLIM). Thus, there is a need to acquire high-speed three-dimensional FLIM images. That was the impetus for the development of the tool.Shown are 3-D fluorescence lifetime imaging microscopy (FLIM) images of pollen grains. Fluorescence intensity (a) and intensity-merged false-color FLIM map (b) for one slice in the image stack. The panel on the right shows a single movie frame from a 3-D rendering of the whole image stack. Scale bars in (a) and (b) are 2.5 μm, and the volume rendered in (c) is 25.6 × 40 × 75 μm.The researchers used a 16-element photomultiplier array from Berlin-based Becker & Hickl GmbH. This device performs time-correlated single-photon-counting detection at each element, a vital ability given the low levels of fluorescence in live-cell studies.The scientists created an array of 16 excitation spots and imaged the resulting fluorescence using a beamsplitter and scanner from LaVision BioTec of Bielefeld, Germany, and an inverted microscope made by Olympus. The foci at the sample were about 2.5 μm apart.For a light source, the investigators used a Ti:sapphire laser from Spectra-Physics of Mountain View, Calif., tuning the output as needed from about 700 to 1000 nm. They scanned the beams in one direction and moved the stage perpendicularly.They captured the fluorescence of a set of 16 lines for times that ranged from half a second to tens of seconds. The integration time depended on the brightness of the sample. After processing the data, they created stacks of FLIM information for imaging or analysis.The researchers examined fluorescently labeled pollen grains and cells transfected to express green fluorescent protein with the system and investigated changes in the fluorescence lifetime of cellular NADH, an important coenzyme found in cells; such changes eventually could be used to identify cancer cells. They measured the autofluorescence from single cells, following metabolic inhibition via NaCN, which blocks a process involving NADH within a cell.Although these measurements were difficult, the scientists detected a change in the fluorescence lifetime for a small number of cells following stimulation. Work is under way to improve upon their results.As for the future, Dunsby said that enhancements could cut the total scan time of a sample. “Further improvements in speed may be realized using improved detector arrays with, for example, higher quantum efficiencies and/or a larger number of channels.” Commercialization, though, might require a different microscope configuration. The present one has both beam and stage scanning, which may be too complex and costly.Optics Express, Oct. 1, 2007, pp. 12548-12561.