Fluorometer with reference light source

Abstract

There is disclosed herein a reference system for a fluorometer designed to detect very low levels of materials tagged with fluorophores. There is also disclosed an optical system for use in such a system which improves the signal to noise ratio. The reference system utilizes pulsed arc light excitation which excitation pulses are directed onto a flow cell containing the fluorescent dye. Fluorescent light emitted from the dye is guided to a photomultiplier tube which converts it to electrical pulses. A portion of each excitation light pulse is guided by a light pipe onto a PIN diode light detector which converts these light signals to electrical pulses. A LED reference light source is pulsed to generate a plurality of reference light pulses one of which occurs between each excitation pulse. A portion of each of these pulses is guided to each of the two light detectors and two more series of electrical pulses are generated. A microprocessor then reads the four electrical pulses resulting from each pair of light pulses and performs a computation on the resulting numbers which indicates the relative concentration of the target concentration being assayed. The optical system makes the excitation light pulses and the emitted light pulses to minimize the amount of scattered excitation light that gets into the emitted light optical channel and to control the location and size of the image projected onto the photomultiplier tube to stabilize its output signal. The light pipe and an output lens spatially integrates the image of the excitation light mask and focusses this light on the PIN diode so that the dancing image of the arc does not wander off the face of the PIN diode and destabilize its output signal. The action of the flow cell fluid contents spatially intergrates the fluorescent light thereby helping to stabilize the output of the photomultiplier tube.