Blue Laser Diode Improves Fluorescence Imaging
Researchers at London's Imperial College of Science, Technology and Medicine have developed a wide-field fluorescence lifetime imaging system that can simultaneously sample up to 36 wells on a multiwell plate. The technique, which employs a blue laser diode as the excitation source, holds promise for rapid imaging in applications such as drug discovery and parallel gene sequencing.
In this image of a rat's ear, fluorescence lifetime imaging using a blue laser diode allows identification of features such as a blood vessel (blue) and elastic cartilage (red). Courtesy of Daniel S. Elson.
Fluorescence lifetime imaging is conceptually straightforward: Excite a fluorophore and watch how long it takes the fluorescence to decay. Typically, such setups combine scanning systems and time-correlated single-photon-counting detection to catch every photon emitted from the fluorophore. This approach, however, tends to be slow because of the time needed for each scan. A time-gated wide-field system speeds up the process, but it runs the risk of inducing photodamage in the sample because several time gates must be used to acquire a usable image.
Daniel S. Elson, lead author of a paper describing the improved technique, said that the team's wide-field system features a picosecond blue laser diode. The laser, which is more commonly used for single-point measurements, has not been applied to wide-field fluorescence lifetime imaging before because the signal level achievable with the diode has been too low, he explained.
The system also incorporates a gated optical image intensifier from Kentech Instruments Ltd. and an intensified 8-bit CCD camera from Photonic Science Ltd. The laser's output is coupled to an optical fiber and reflected off a mirror to the underside of a standard multiwell plate. The fluorescence emitted from the wells is reflected again from the mirror and through a filter that blocks any excitation light that was also reflected. The emission is focused on the gated optical image intensifier, and the CCD camera records the images from the intensifier.
The researchers tested the system on several combinations of fluorophores and solvents and found that it can image the nonradiative decay by environmental perturbations and can resolve lifetime differences as short as 50 ps. They also tested the blue laser in a fluorescence lifetime microscope setup. Unlike conventional mode-locked solid-state lasers, the laser diode's repetition rate can be adjusted from 0 to 40 MHz. The ability to adjust the repetition rate should enable the user to choose an appropriate interpulse spacing for the lifetime under measurement.
Applying the technique to a commercial multiwell plate reader could prove useful for the parallel investigation of an organism's genome or for comparing the expression of particular genes in different samples, Elson said.
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