Two light source reference system for a fluorometer

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
• RFI=(A/B).times.(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.

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.

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
• A/B
• and multiplying it by the ratio
• C/D.