Two light source reference system for a fluorometer

• US 4,802,768 A
• Filed: 04/11/1986
• Issued: 02/07/1989
• Est. Priority Date: 04/11/1986
• Status: Expired due to Fees

• Current Assignee: Sclavo SPA
• Original Assignee: Sclavo SPA
• Inventors: Gifford, Charles R., Bigliardi, Achille M.
• Primary Examiner(s): McGraw, Vincent P.
• Application Number:
  US06/850,944
• Time in Patent Office: 1,033 Days
• Field of Search: 356/301, 356/317, 356/318, 356/328, 356/319, 356/246, 356/435, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/467.2, 250/458.1
• 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 2201/0696 Pulsed
• CPC Class Codes: G01N 2201/0698 Using reference pulsed source
• CPC Class Codes: G01N 2201/082 Fibres for a reference path

Abstract

A reference system utilizes pulsed arc light excitation which excitation pulses are directed onto a flow cell containing a 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. An optical system masks 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 spatially integrates the images of the arc yielding the excitation light to control the stability of the image projected onto the photomultiplier tube. The light pipe and an output lens spatially integrates the image of the excitation light mask and focuses this light on the PIN diode so that the dancing image of the arc does not modulate with the variations of the PIN diode cathode and destabilize its output signal.

Claims

1. An apparatus comprising:

an arc light means having electrodes, for exciting a variable unknown and very small quantity of fluoroescent dye chemically attached to a material being assayed with intense excitation pulses of light generated from an arc occurring between said electrodes, said excitation light pulses having at least some wavelengths in the excitation band of said fluorescent dye;

flow cell means for containing said material to which said fluorescent dye molecules are attached, said flow cell having a flat, transparent input window of quartz glass having a shape which is longer in one dimension than the other, said longer dimension having an axis which is parallel to the axis between said electrodes in said arc light means between which said arc appears, said flow cell also having a flat, transparent exit window of quartz glass having a normal which is at right angles to the normal of said input window, said exit window being the path through which fluorescent light emitted by excited dye molecules travels, said flow cell having at least the intersection of said input window and said exit window formed by opaque material so as to block the path of scattered light traveling inside the input window toward said exit window by refraction between the quartz surfaces thereby preventing such scattered light from entering the paths used by light exiting from said exit window;

first mask means in the optical path between said arc light means and said input window for limiting the image of the arc from wandering on the surface of said flow cell outside the bounds of said input window;

photomultiplier tube means for detecting fluorescent light emitted from said dye in response to said excitation light pulses and for generating signals indicting the intensity of said light emitted from said dye;

means for focusing emitted fluorescent light on said photomultiplier tube means and for allowing only light rays emanating from the interior of said flow cell means and having a frequency in the band of fluorescent light frequencies emanating from excited dye molecules to reach said photomultiplier means and for preventing stray excitation light scattered from the walls of said flow cell means from following the path traveled by said fluoroescent light rays emanating from the inside of said flow cell means;

reference light detector means for detecting the intensity of light pulses shining on a light sensitive surface and for generating an output signal indicating said intensity;

light waveguide means for coupling light from said arc to said reference light detector means and for spatially integrating the image of said arc so that there is no arc image that wanders randomly over said light sensitive surface of said reference light detector means;

reference light source means for supplying pulses of reference light to each of said photomultiplier tube means and to said reference light detector means during the dark intervals between excitation light pulses; and

means for calculating a relative fluorescence index from the output signals from said reference light detector means in response to both said excitation light pulses and said reference light pulses and from the output signals from said photomultiplier tube means in response to both emitted fluorescent light pulses and reference light pulses, said relative fluorescence index indicating the amount of fluorescent light being emitted from said flow cell and being compensated for changes in intensity of either the reference light pulses or the excitation light pulses and being compensated for changes in the sensitivity of either said photomultiplier tube means or said reference light detector means.

2. Apparatus as defined in claim 1 wherein said flow cell means includes a mirrored surface opposite said input window to reflect excitation light pulses back through said dye and a mirrored surface opposite said exit window for reflecting emitted fluorescent light rays traveling away from said exit window toward said exit window.

3. Apparatus as defined in claim 1 wherein said flow cell means includes means for containing said dye in a confined space with a geometry which is longer in one dimension than the other.

4. Apparatus as defined in claim 3 further comprising means for spatially integrating the image of said arc on said dye.

5. The apparatus of claim 1 wherein said means for calculating comprises:

means for calculating the ratio

space="preserve" listing-type="equation">(A/B)×

(C/D) where

A=the magnitude of the output signal from said photomultiplier tube means generated in response to receipt of fluorescent light from said dye emitted in response to excitation of said dye by an excitation light pulse, and

B=the magnitude of the output signal from said reference light detector means in response to receipt of a portion of the same excitation light pulse which caused response A after being spatially integrated by said light waveguide means, and

C=the magnitude of the output signal from said reference light detector means in response to receipt of a reference light pulse from said reference light source means, and

D=the magnitude of the output signal from said photomultiplier tube means generated in response to receipt of the same reference light pulse from said reference light source means that caused response C.

6. The apparatus of claim 5 wherein the terms A, B, C and D are as defined less the magnitude of the response of the respective detecting means when not receiving a light pulse.

7. The apparatus as defined in claim 5 wherein said eight waveguide means is a light pipe.

8. The apparatus of claim 1 wherein said flow cell means has a geometry which is selected to conform to the general shape of the volume through which said arc wanders.

9. A reference system for a fluorescence measuring system having a flow cell containing fluorescent dye having an excitation band of frequencies and an emission band of frequencies, a first light source having an arc between two electrodes for illuminating said dye in said excitation spectrum, and a first light detector for detecting fluorescent light in said emission spectrum emitted from said dye comprising:

means for causing said first light source to emit multiple pulses of light separated by dark intervals;

means for reducing the amount of light in said excitation spectrum light pulses from said first light source which is scattered to said first light detector and for maximizing the amount of fluorescent light emitted in said emission spectrum which reaches said first light detector comprising transparent excitation and exit light windows in said flow cell which have faces which are at right angles to each other, said flow cell comprising a chamber having a rectangular shaped cross-section and having opaque corners to cut off the path of light rays traveling in said excitation light window toward said exit light window, said excitation light window having a long dimension and a short dimension of a transparent portion, said long dimension parallel to the line between said electrodes, and further comprising a lens, a mask means and filter means between said arc and said excitation light window, said mask means for limiting an image of said arc created by said lens from wandering outside said transparent portion, said filter means for reducing the amplitude of any light from said arc in the emission spectrum which reaches said flow cell;

a second light detector;

means for diverting a portion of each said

pulse from said first light source to said second light detector;

a second light source;

means for causing said second light source to emit multiple pulses of light during the dark intervals of said first light source;

means for directing a portion of each said pulse of light from said second light source to each of said first and second light detectors;

means for quantifying the signals from said first and second light detectors in response to said light pulses from said first and second light sources; and

means for calculating the ratio

space="preserve" listing-type="equation">RFI=(A/B)×

(C/D)

where

A=the response of said first light detector to fluorescent light emitted from the dye in said flow cell emitted in response to a pulse from said first light source less the response of said first light detector in the absence of any said pulse or other light;

B=the response of said second light detector to a portion of the same said light pulse from said first light source which caused response A less the response of said second light detector in the absence of any said pulse or other light;

C=the response of said second light detector in response to a light pulse from said second light source less its response in the absence of said pulse or any other said light; and

D=the response of said first light detector in response to the same light pulse from said second light source that caused response C less its response in the absence of said pulse or any other light, and RFI=the relative fluorescence index of said dye.

17. An apparatus as defined in claim 9 wherein said flow cell includes a mirrored surface opposite said excitation light window to reflect excitation light pulses back through said dye and a mirrored surface opposite said exit light window for reflecting emitted fluorescent light rays traveling away from said exit light window toward said exit light window.

10. An apparatus comprising:

a flow cell containing a material being assayed, said material being tagged with a fluorescent dye and having a transparent input window for allowing excitation light to impinge on said dye and a transparent output window for allowing fluorescent light emitted in response to said excitation light to exit;

an excitation arc light source for generating excitation light pulses from an arc having a frequency in the excitation spectrum of said dye;

a first optical path means for guiding said excitation light pulses and focusing the image of said arc onto the dye inside said flow cell through said transparent input window, and for filtering out any light not in the excitation spectrum of said dye from entering said flow cell;

means in said first optical path for limiting the light energy in the image of said arc to an area within a predetermined area on the face of said flow cell having a geometry generally conforming to the geometry of said transparent input window;

means for spatially integrating the image of said arc in said dye;

a photomultiplier tube light detector having a light input;

a second optical path for guiding emitted fluorescent light emerging from said transparent output window to the light input of said photomultiplier tube and for focusing the emitted fluorescent light onto the light sensitive surface of said photomultiplier tube and for filtering any light not in the frequency band of said emitted fluorescent light and for limiting the shape and size of the image of said flow cell projected onto the light sensitive surface of said photomultiplier tube;

a beam splitter in said first optical path for diverting a portion of each said excitation light pulse to an output;

a light pipe having an optical input optically coupled to said output of said beam splitter and having an optical output;

a second light detector having a light sensitive surface located such that light emerging from the output of said light pipe falls upon said optical input of second light detector;

a lens interposed between said light output of said light pipe and the light sensitive surface of said second light detector for capturing light emerging from said light output and focusing it upon said light sensitive surface of said second light detector;

a reference light source of generating reference light pulses which occur during dark intervals between excitation light pulses; and

a third optical path for guiding a portion of each reference light pulse to said light sensitive surface of said second light detector and a portion of each reference light pulse to the light sensitive surface of said photomultiplier tube.

11. The apparatus of claim 10 further comprising means coupled to said photomultiplier tube and to said second light detector for calculating the relative intensity of fluorescent light emitted from said flow cell in a manner which yields a result which is substantially independent of all changes in the system except changes in the amount of dye molecules in said flow cell.

12. The apparatus of claim 11 wherein said means for quantifying includes means for calculating a first ratio equal to the response of said photomultiplier tube to each excitation light pulse divided by the response of said second light detector to each excitation light pulse and for multiplying said first ratio by a second ratio equal to the response of said second light detector to each reference light pulse divided by the response of said photomultiplier tube to each said reference light pulse.

13. The apparatus of claim 12 further comprising means for subtracting from each response of each term in the numerator and the denominator of each said ratio, the response of the respective detector in the absence of any light pulse.

14. A process for quantifying the amount of fluorescent dye in a flow cell comprising the steps of:

exciting the dye with an excitation light pulse from an arc which wanders in space between two electrodes, said light from said arc having been focused, and passband filtered so that only excitation light frequencies impinge on the dye and said arc light having been passed through a mask of predetermined geometry such that said arc light is masked such that the wandering of the image of the arc on the dye is limited to within a known geometric area;

guiding the resulting emitted fluorescent light to a first light detector with a lens, and a passband filter and a mask to focus the emitted fluorescent light in a beam, filter out light rays having a frequency other than the frequency of the emitted fluorescent light and to limit the geometry of the image of the flow cell on the light sensitive surface of the first light detector;

calculating the amount of fluorescent light emitted from said dye in response to said excitation light pulse using said first light detector and storing it as a variable A;

guiding a portion of said excitation pulse to a second light detector and spatially integrating the image of the excitation light source in the process;

calculating the amount of light from said excitation light pulse falling upon said second light detector and storing it as a variable B;

generating a reference light pulse;

guiding a portion of said reference light pulse to each of said first and second light detectors;

calculating the amount of said reference light pulse falling on said second light detector and storing it as a variable C;

calculating the amount of said reference light pulse falling on said first light detector and storing it as a variable D; and

calculating the ratio

space="preserve" listing-type="equation">A/B

and multiplying it by the ratio

space="preserve" listing-type="equation">C/D.

15. The method of claim 14 further comprising the steps of calculating the amount of light falling on said first light detector in the absence of any light pulse and storing it as the variable Z1 and calculating the amount of light falling on said second light detector in the absence of any light pulse and storing it as the variable Z2 and wherein the last step in the method of claim 24 is modified to calculate

16. The method of claim 15 further comprising the step of blocking the transmission of substantially all of said excitation pulse to said first detector so that the variable A indicates substantially only the amount of fluorescent light emitted from said dye in response to said excitation pulse.

18. An apparatus as defined in claim 14 further comprising means in the light path between said flow cell and said first light detector for filtering out any light not having a frequency in the frequency band of fluorescent light emitted by said dye and for focusing emitted fluorescent light on said first light detector and for limiting the extent of the image of said exit light window on the light sensitive surface of said first detector.