A Compact Violet Diode Laser-Based Fluorescence Lifetime Microscope

We have constructed a time-correlated single-photon counting (TCSPC) microscope system using a pulsed violet-laser diode for measuring fluorescence lifetimes below 1 ns. These compact; cool, and fast-pulsed laser diodes are much more suitable for lifetime measurements than the traditional mode-locked lasers used in the past. The laser source (LDH-400, PicoQuant GmbH) is mounted on an optical table above the microscope, and the laser beam is directed into an Olympus BX-60 microscope by means of a dichroic beamsplitter set at 45 degrees. The system operates in backscattering mode with the fluorescence emission passing back through the dichroic beamsplitter and focused into a 100 mm focal length monochromator with a PMT detector. This prototype instrument is compact (∼80 × 70 × 70 cm) and is nearly fully computer controlled by means of a SPC-730 (Becker & Hickl) PC card. We include preliminary results showing the instrument response function (IRF) of the system, and some of the factors have been adjusted to minimize the temporal width of the IRF. The instrument has been validated using a series of standard fluorophores at different emission wavelengths.