Optics: measuring and testing – Photometers
Reexamination Certificate
1997-05-09
2001-02-13
Font, Frank G. (Department: 2877)
Optics: measuring and testing
Photometers
Reexamination Certificate
active
06188473
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a method and system therefor for photodetection in which measurement signals in the form of signal pulses during a photon-counting operation are tapped off at a photomultiplier, the signal pulses being indicative of incident photons of the light being measured. Methods, systems and devices therefor which employ such photodetection schemes are particularly useful in chemical engineering, especially for performing highly sensitive analyses based on the principle of chemoluminescence or bioluminescence, such as luminescence immunoassays for example, reporter gene assays, or DNA assays. For such purposes, it is necessary to record the luminescence process, which occurs as a rule in the seconds range, with time resolution, with a high dynamic range being required for detecting the light quanta to investigate samples of an unknown concentration. In this connection, it is important to note that samples, as a rule, are investigated automatically at a high throughput rate and that they can only be measured once after the luminescence reaction has started.
To guarantee a high detection sensitivity, for example, in a known photodetection system for performing high sensitivity analyses based on the principle of chemoluminescence or bioluminescence, the photomultiplier that serves as the opto-receiver is employed in a so-called single-photon counting operation. In such operation, the photomultiplier provides sufficient amplification because of the presence of the selected high voltage and the many dynode stages to cause the generation of pulses of current or voltage that can effect the triggering of a single photoelectron. The voltage pulses thus produced can be distinguished from background noise by a discriminator. Depending on the quantum yield of the photocathode, therefore, individual incident photons can be detected. The disadvantage of this process, however, is that the bandwidth of known pulse amplifiers is insufficient at higher light intensities to resolve individual events in time without dead time losses. At higher photon rates, so-called pile-up effects cause problems, due to an overlapping of pulses.
It is also known in conjunction with the operation of photomultipliers to amplify the output signals in analog fashion in the so-called current mode, in which the detection sensitivity is reduced by suitable adjustment of the operation voltage, although higher light intensities can still be measured. The disadvantage of this operating mode, in addition to a reduced sensitivity, lies particularly in the low long-term stability caused primarily by changes in the quantum efficiency of the photocathode and multiplication fluctuations that result from dynode fatigue and hysteresis.
SUMMARY OF THE INVENTION
It is a main objective of the present invention to realize an improved photodetection method and a system therefor employing such method which performs high sensitivity analysis such that light measurements with a high degree of accuracy are achievable over a wide dynamic range.
This as well as other objectives are realizable according to a method and system therefor of the invention, defined in the appended claims, from the brief description hereinbelow as well as from the detailed description of the illustrated embodiment (although not limited thereto).
Briefly, the invention calls for wiring the photomultiplier with two channels, with one channel operating as a photon counter which responds to the pulsing action effected by the signal pulses tapped at the photomultiplier and the other channel operating in an analog mode for detecting higher light flux values. That is, the invention proposed in accordance with the improved method and system therefor, calls for a current signal, triggered by the photons, to be derived at the photomultiplier parallel to the signal pulses and the measured value for the light to be measured during each of the time intervals is determined from the number of pulses below a range limit that can be set, and from the current signal above the range limit. To permit automatic range selection, the range limit is advantageously set to have a predetermined upper limiting value for the number of pulses. It is advantageous, in this connection, for the upper limiting value to be determined from a pulse count range in which the photomultiplier can be operated as a photon counter at a given operating voltage. To process the current signals, also, it is advantageous for a current integrated measurand to be formed during each of the time intervals from the current signal amplified in analog fashion.
According to one preferred embodiment of the invention, a correction factor for drift compensation of the current integrated measurand is determined at predetermined time intervals from the pulse number and the current integrated measurand associated therewith, in time. This makes it possible to utilize the pulse count that can be performed on the basis of noise signal discrimination with high accuracy to compensate for long-term drift of the analog current signal. This can be accomplished by the correction factor being determined as a quotient of the measured number of pulses and the current integrated measurand associated therewith, timewise.
Advantageously, the photocathode of the photomultiplier is illuminated at predetermined time intervals by a pulsed calibration light source, preferably designed as a light emitting diode (LED), to determine the correction factor. The possibility of adjusting the intensity of the light source allows the photomultiplier to be operated at a working point that is suitable for the photon-counting operation as well as for current operation.
Another improvement can be accomplished by monitoring the functional readiness of the photomultiplier during illumination by the calibrating light source, with the number of pulses being compared with an anticipated value. In this fashion, at least the theoretical function of the photomultiplier as a photon counter can be checked.
As an alternative to using a calibration light source, the correction factor can be determined using the light to be measured. For this purpose, the correction factor is determined at time intervals during which the number of pulses is between the upper limiting value and a lower limiting value, with the photomultiplier being operated up to the lower limiting value in current operation and up to the upper limiting value in photon-counting mode.
Advantageously, zero point drift can be compensated by determining an offset compensation value from the current integrated measurand or the current signal during dark measurements repeated at time intervals. In this way, the drift-compensated measured value for the light to be measured can be calculated by multiplying the difference between the current integrated measurand and the offset compensation value with the correction factor just determined. It is also advantageous, when a predetermined threshold value is exceeded for the current signal or the current integrated measurand, that the operating voltage of the photomultiplier be reduced automatically in order to reduce the risk of damage to the detector. Advantageously, a measured value obtained at a reduced operating voltage is rejected as invalid.
From the circuit standpoint, it is advantageous for the current signal to be derived through a resistor from the first or second dynode stage, located downstream from the photocathode of the photomultiplier. If the photocathode is at ground potential, a signal can be conducted at a low potential level to the amplifier. The signal pulses can be tapped off advantageously through a capacitor to unblock the high voltage at the anode of the photomultiplier.
To permit digital processing, the current integrated measurand can be formed by effecting voltage-to-frequency conversion of the current signal into a proportional pulse frequency and adding the pulses thus obtained during each time interval. It is also advantageous for the number of pulses obtained at intervals during a measuring pha
Eppler Jorg
Leistner Hermann
Retzlaff Rüdiger
Antonelli Terry Stout & Kraus LLP
Font Frank G.
Nguyen Tu T.
Stratec Electronik GmbH
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