Journal of Fluorescence, Vol. 12, No. 2, June 2002 ( 2002) A Compact Violet Diode Laser–Based Fluorescence Lifetime Microscope Alan G. Ryder, 1,2 Thomas J. Glynn, 1 Milosz Przyjalgowski, 1 and Boguslaw Szczupak 1 Received November 23, 2001 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 backscatter- ing 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. KEY WORDS: Fluorescence; lifetime; laser diode; microscopy. INTRODUCTION domain measurement methods, have been reported, although most of these instruments incorporate sophisti- cated laser systems as the excitation source, which require Fluorescence microscopy is an important measuring high maintenance and make also them expensive to tool in the life sciences because of its inherent sensitivity acquire [3–5]. Semiconductor laser diodes offer the and the development of very specific fluorescent probes. advantage of small size and low costs, and recently a The majority of fluoresce microscopy systems in use tend TRFM system based on a 635-nm diode has been to be based on steady-state techniques, partly due to cost reported [6]. and technology issues. Steady-state systems, however, We present a TRFM system based on a pulsed violet suffer from problems such as photobleaching, probe con- laser diode using the TCSPC method. This is a simple centration variation, and scattering artifacts that affect and versatile system primarily intended for analysis of fluorescence intensity measurements [1]. time-resolved microscopic petroleum fluid inclusions and biological fluorescence microscopy (TRFM) systems, on the other systems. hand, are much less sensitive to these effects [2]. Systems for TRFM, which usually utilize either frequency or time INSTRUMENTATION 1 Department of Physics, National University of Ireland, Galway, Ireland. The microscope system is depicted in Fig. 1 and 2 To whom correspondence should be addressed. Tel: 353-91-750469. Fax: 353-91-750584. Email: alan.ryder@nuigalway.ie consists of a 402-nm violet laser diode, which can be 177 1053-0509/02/0600-0177/0  2002 Plenum Publishing Corporation