Sep 4, 1973 - and Bell, Norwood,. Ohio 45212) in water. LD standards were prepared from a 50 U/mi stock. (Sigma) diluted with the KH2PO4 buffer described.
CLIN. CHEM.
19/11, 1259-1264 (1973)
Design and Evaluation of a Filter Fluorometer That Incorporates a Photon-Counting Detector Robert E. Curry, Harry L. Pardue,1 Glen E. Mieling, and Robert E. Santini
We describe the design and evaluation of a filter fluorometer in which a photomultiplier detector is used in the photon-counting mode. Output frequency is shown to be linear with light intensity from 5 Hz to 800 kHz. Comparisons with commercially available instruments show an inherent sensitivity superior to a commercially available filter instrument and comparable to that of a spectrofluorometer. Results are included showing the applicability of the instrument to the kinetic determination of lactate dehydrogenase in serum. Additional Keyphrases: lactate rescein
#{149}rhodamine
B
dehydrogenase sensitivity
#{149} tluo-
#{149}improved
One factor that limits the ultimate sensitivity of any spectroscopic method involving measurement of low light intensities is the background signal resulting from detector dark-current noise. Recent reports demonstrate that photon counting is an effective method for minimizing dark-current contributions in photomultiplier tubes (1-3). The improved dark-current rejection results from the fact that electrons emitted from the dynodes are amplified less than electrons emitted at the photocathode, and leveldiscriminating circuitry can be used to differentiate between the dark current and photon signals. Analytical procedures based on fluorescence are recognized as being among the most sensitive spectroscopic methods (4). Because these methods are invariably based on the measurement of low light intensities, photon counting would probably be useful in extending the lower limits of sensitivity. The work reported here was undertaken to evaluate the relative performance characteristics of a relatively simple, inexpensive photon-counting fluorometer and the more conventional fluorometers that operate in the analog mode. This report includes design details of a photon-counting filter fluorometer, fundamental information that influenced decisions on several design options, quantitative comparisons between the photon-counting instrument and commercially available filterand spectrofluorometers, and analytical results for the fluorometric determination of lactate dehydrogenase (EC 1.1.1.27). From the Department of Chemistry. Purdue University, ette. Ind. 47907. 1 Correspondence should be addressed to this author. Received April 30, 1973, accepted Sept. 4. 1973.
Lafay-
Materials
and Methods
Apparatus Our primary goal of evaluating the performance characteristics of a photon-counting detector system for fluorometric analyses dictated an excitation source with stability characteristics well beyond those expected of the detector system. Since xenon and other arc sources tend to be unstable, we chose to sacrifice ultraviolet response in favor of the high stability that can be achieved from filament-type lamps. A high-wattage tungsten halogen lamp, driven by a regulated power supply, was used to provide the high stability excitation source required in this study. The optical system is represented in Figure 1. The system is constructed on a 1/4-inch stainless-steel plate mounted on 2-inch high steel strips. The excitation source is a 1000-W tungsten-halogen lamp (Type EGJ; Sylvania Electric Products, Inc, Danvers, Mass. 01923) mounted firmly on the support plate and operated at 700 W (7 A Li 100 V dc). The lamp is enclosed in a housing constructed from two brass cylinders such that coolant (tap water) can be circulated near the lamp. A lens having a focal length of 4.5-cm (of ordinary-grade glass from Edmund Scientific, Barrington, N. J. 08007) is placed at the exit port of the lamp housing. A beam splitter (M.P. 1022; McKee-Pedersen Instruments, Danville, Calif. 94525) is located with its center at the focal point of the lens. A second lens with focal length of
f.-d2
-
d3
+
d4H
II Fig.
1.
Optical layout of the fluorometer
9.0 cm: d3 = 9.0 cm: d4 = 7.5 cm: d5 = 7.0 cm: d6 = 2.5cm J, Brass cooling jacket for source: S. Tungsten halogen lamp (Sylvania EGJ-1000W); LI, Glass lens (4.5 cm focal length); L2, Glass lens (4.0 cm focal length): BS, Beam splitter (McKee-Pederson); PT. Phototube (RCA 935); Fl, Excitation filter; F2, Emission filter: C, Quartz cell (1 cm): CH, Sample cell housing (Mcl
