Fluorescence lifetime imaging microscopy (FLIM) can make quantitative measurements when other techniques cannot. For example, FLIM can discriminate among multiple fluorophores, even in turbid backgrounds such as tissues and cells. However, detecting these subtle differences requires imaging equipment with high temporal resolution. A new high-resolution time-gated CCD camera can distinguish among fluorophores with nanosecond lifetimes, and it can separate fluorophores from a confounding turbid fluorescent medium.In the past, researchers have recorded lifetime images using image-intensified cameras or time-correlated single-photon counting boards. However, intensified cameras produce noisy images for various reasons, including the heterogeneity of the intensifier components. In contrast, a time-gated CCD does not require additional parts that increase noise. And time-correlated single-photon counting boards can measure only the total fluorescence lifetime, whereas time-gated CCD cameras also can measure portions of the fluorescence lifetime. These time-gated measurements can enable FLIM with multiple labels with similar lifetimes, and it can allow discrimination among longer-lived fluorophores and shorter-lived lifetime signals such as those caused by autofluorescence. Christopher G. Morgan and colleagues developed the CCD camera. Development began at the University of Salford in the UK and continued at Photonic Research Systems Ltd., a spin-off company also located in Salford. The researchers created a prototype color imager with a complementary color mask and accompanying software.The camera employs a Peltier-cooled Sony interline CCD with a charge drain that prevents its collecting light when not activated. Synchronization of the charge drain to pulsed laser excitation enables nanosecond time gating, and the camera can integrate many laser pulses before readout. The software and camera hardware incorporate features designed so that novices can perform FLIM.For the excitation source, the researchers employed a Q-switched Nd:YVO4 laser from Elforlight of Daventry, UK. The laser delivered nanosecond pulses with a repetition rate that varied from about 1 to 20 kHz. To remove spots that coherent emission produces, the laser beam traveled through a fiber connected to a vibration unit from Technical Video of Port Townsend, Wash. The researchers obtained 532-nm (green) and 355-nm (UV) wavelengths with optical filters from Edmund Optics and Glen Spectra, and the camera was fitted with a filter to block scattered excitation.Figure 1. The researchers used the camera to capture, through a confounding turbid fluorescent background, these images of quantum dots deposited on a cross. The first frame was taken with a 50-ns delay, and each subsequent frame was taken in 10-ns increments.In their first experiment, they deposited quantum dots on a paper cross on top of which they placed a beaker full of a turbid fluorescent medium, completely obscuring the quantum dots. They attempted to image long-lived quantum dot emission through the fluorescent liquid. In time-gated mode, the CCD camera detected the paper cross through the medium (Figure 1). Morgan presented the lifetime images at Photonics West in January. In his talk, he demonstrated that the camera can image fluorophores with lifetimes down to 0.5 ns.In their second experiment, the researchers captured true-color, time-gated images of sulforhodamine B, fluorescein and quinine sulfate with sodium chloride added to reduce its fluorescence lifetime. In addition to the color time-gated images, they obtained fluorescence lifetime images. The fluorophores have lifetimes of 2, 4 and 8 ns, respectively. The uniform intensity of each fluorophore in its respective cuvette shows that the resulting fluorescence lifetime images have a high signal-to-noise ratio (Figure 2). Figure 2. With a novel CCD camera, the researchers recorded true-color time-gated images of three fluorescent labels with different lifetimes and emission spectra (a, b and c). They also used the camera to take conventional fluorescence lifetime images (d). The image is pseudocolored and was processed to add intensity information. The uniformity of the color intensity shows that the image has a high signal-to-noise ratio.Morgan said that they took the measurements at least five times in both experiments and that they are reproducible. The researchers also are using the color CCD camera to record Förster resonance energy transfer images. Morgan said that they are examining whether a long-lifetime lipophilic energy donor (pyrene-labeled decanoic acid) and a short-lifetime energy acceptor (octadecyl rhodamine B) bind jointly to detergent micelles. They are experimenting with fitting various red-green-blue filters to the camera so that they can achieve the best spatial and spectral resolution for fluorescence lifetime imaging.