Fluorometer with reference light source

• US 4,750,837 A
• Filed: 04/11/1986
• Issued: 06/14/1988
• Est. Priority Date: 04/11/1986
• Status: Expired due to Fees

• Current Assignee: Sclavo SPA
• Original Assignee: Sclavo SPA
• Inventors: Bigliardi, Achille M., Gifford, Charles R.
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US06/850,933
• Time in Patent Office: 795 Days
• Field of Search: 356/301, 356/317, 356/318, 356/319, 356/328, 356/246, 356/435, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2
• US Class Current: 356/417
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 21/255 Details, e.g. use of specially adapted sources, lighting or optical systems
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2201/064 Stray light conditioning
• CPC Class Codes: G01N 2201/0642 Light traps; baffles
• CPC Class Codes: G01N 2201/0696 Pulsed
• CPC Class Codes: G01N 2201/0698 Using reference pulsed source
• CPC Class Codes: G01N 2201/082 Fibres for a reference path

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.

Claims

1. A fluorometer comprising:

a flow cell containing sample tagged with a fluorophore and having input and output windows made of clear quartz, a generally rectangular cross section, a shape which is longer than it is wide, and opaque corners forming each of the four corners in the rectangular shaped cell;

a first light detector;

a first pulsed light source means for generating a plurality of excitation light pulses;

a first optical path means for guiding said excitation light pulse to an input window of said flow cell;

a first mask means in said first optical path for limiting the size of the image of said pulsed light source means which is focused in said flow cell such that substantially no light rays forming said image fall upon any portion of said flow cell outside said input window;

a second optical path means for guiding emitted fluorescent light from said fluorophore to said first light detector;

a second mask in said second optical path means for limiting the geometry of the image of the output window of said flow cell such that the image is completely contained by the light sensitive surface of said first light detector;

an optical passband filter means in said second optical path means for allowing transmission therethrough of emitted fluorescent light from said dye but for substantially blocking transmission of light of wavelengths other than the wavelengths of said emitted fluorescent light;

a second light detector;

a third optical path means for conducting a portion of the excitation light to said second light detector;

a second pulsed light source means for generating a plurality of reference light pulses during a dark interval between excitation pulses; and

a fourth optical path means for guiding a portion of each reference light pulse onto each of said first and second light detectors.

2. The apparatus of claim 1 wherein said flow cell has mirrored surfaces opposite said input and said output windows.

3. The apparatus of claim 1 wherein said flow cell has an internal cavity with a rectangular cross section and is fed from a hose having a circular cross section, said apparatus further comprising end caps for guiding sample aliquots and air pegs into and out of said flow cell, said end caps making a transition from the round shape of the hose to the rectangular shape of the internal cavity of said flow cell.

4. The system of claim 1 wherein said excitation light and reference light are of different frequencies, said system further comprising means for filtering the portion of said reference light pulse guided toward said first light detector so as to substantially block transmission of any excitation light from said third optical path to said first light detector.