Simple Time-Resolved Fluorometer Based on a Nanosecond Digital Oscilloscope and a Diode-Pumped, Solid-State Laser FRANCO BASILE, ANITA CARDAMONE*, KEITH D. GRINSTEAD, JR., KAREN J. MILLER,. FRED E. LYTLE,t ANDREA CAPRARA, CHRIS D. CLARK, and JEAN-MARC HERITIER Department of Chemistry, 1393 Brown Laboratories, Purdue University, West Lafayette, Indiana 47907-1393 (F.B., A.C., K.D.G., K.J.M., F.E.L); and Continuum, Inc., 3150 Central Expressway, Santa Clara, California 95051 (A.C., C.D.C., J.-M.H.) A simple time-resolved fluorometer is constructed with an all-solid-state- based, frequency-tripled, Q-switched, diode-pumped Nd:YLF laser as the excitation source. Signal processing is accomplished with a digital oscilloscope. Simplicity of operation and applicability to trace analysis and in time-resolved spectroscopy are demonstrated with this new in- strument. The laser produces 2.5-ns pulses at 349 nm and is capable of kilohertz repetition rates. For every shot of the laser, the oscilloscope collects an entire fluorescence decay at a l-ns digitizer resolution and can average these data at the maximum laser repetition rate. When one is operating at 1 kHz and signal averaging for one second, detection limits (S/N = 3) in the I0-I00 pM region are obtained. Excited-state decays are collected for several enzymatic probes and quinine sulfate, providing lifetimes consistent with those obtained by established instru- ments. Index Headings: Fluorescence; Instrumentation, fast digital oscilloscope; Lasers, diode pumped. INTRODUCTION Methods based on fluorometric analyses of molecules in solution possess the advantage of excellent specificity and sensitivity. However, it has been recognized that fluorometry is blank limited I and that detection limits are often determined by Rayleigh and Raman scatter from the sample matrixY Moreover, it is well known that the incorporation of a laser as the excitation source does not lead to the predicted 4 to 5 order-of-magnitude im- provement in the detection limit2 Since the blank components are mostly short lived (< 5 ns), the combination of pulsed laser excitation and time resolution has produced instrumentation capable of quantitation in the subnanomolar range. 3-5 This capa- bility, combined with a recent surge of applications using excited-state lifetimes as probes of molecular environ- ment, has resulted in an increase in the number of lab- oratories interested in making time-resolved fluoromet- ric measurements2 However, pulsed lasers, or even lasers in general, are not yet widespread as a standard light source for fluorometry in clinical, environmental, and industrial laboratories. This situation is primarily due to the maintenance cost and the need for highly skilled operators to keep the equipment operating at its highest performance. These factors are hindering the dissemi- Received 1 October 1992. * Undergraduate research participant, Department of Chemistry, Ju- niata College, Huntingdon, PA 16652. t Author to whom correspondence should be sent. nation of this technology and methodology to scientists in many fields. What is needed is a simple, reliable, user- friendly pulsed laser combined with a signal processing system familiar to potential users. Diode lasers show many promising features that can make them the excitation source of choice for applica- tions in fluorometry.7 They are inexpensive, and simple to operate and have a relatively long mean-time between failure. Their red to near-infrared outputs forced pre- vious researchers to develop new IR fluorophores. How- ever, diode lasers can be used as the pump source to drive either Nd:YAG or Nd:YLF lasers--which, by fre- quency doubling or tripling, can produce green or ultra- violet wavelengths. As an added benefit, these devices are constructed with rugged all-solid-state components integrated into small geometries. This factor allows the production of highly efficient, compact, and rugged mi- crolasers with more than 10% efficiency s (electrical to optical). Diode lasers have a lifetime ranging from 20,000 to 40,000 hours, decreasing instrument down-time. Moreover, these lasers are powered with less than 5 volts, in comparison with more than 1000 volts for many gas- discharge lasers. An approximate fivefold decrease in the size of the power supply is obtained as a consequence of the low voltages required. This benefit can allow ease in instrument handling and transportation, making the de- vice a more reliable source of coherent light for field work and automated tasks. In an effort to demonstrate the existence of the above advantages, a prototype all-solid-state, frequency-tri- pled, Q-switched, diode-pumped Nd:YLF laser has been constructed. The device has repetition rates in the low- kilohertz domain and a pulse width of 2.5 ns FWHM. It is user friendly, in that no gas supplies or water cooling systems are needed. The unit requires no adjustment of mirrors or current sources prior to use, and takes only 15 min to reach thermal equilibrium. This paper de- scribes a simple time-resolved fluorometer based on the above diode-pumped laser and a 1 GSample/s oscillo- scope to monitor the photomultiplier anode current. The oscilloscope is triggered by an electronic signal from the diode laser power supply, further simplifying this in- strument in comparison to those necessitating an optical timing reference. Calibration curve figures of merit and excited-state lifetimes are reported for molecules used as tags in ami- nopeptidase and ~-galactosidase enzymatic assays, and Volume 47, Number 2, 1993 0003-7028/93/4702-020752.00/0 APPLIED SPECTROSCOPY 207 © 1993Societyfor AppliedSpectroscopy