Fluorometer with low heat-generating light source

Abstract

This invention concerns a fluorometer preferably combined with a thermal cycler useful in biochemical protocols such as polymerase chain reaction (PCR) and DNA melting curve analysis. The present fluorometer features a low heat-generating light source such as a light emitting diode (LED), having a one-to-one correspondence to each of a plurality of sample containers, such as capped PCR tubes in a standard titer tray. The fluorometer of the present invention further comprises an optical path between each LED and its to correspondingly positioned container, and another optical path between each fluorescing sample within the positioned container and an optical signal sensing means. The instrument can be computer controlled.

1 . A fluorometer, comprising:
 a plurality of low heat-generating light sources;   means for positioning a plurality of containers for containing potentially fluorescing sample into optical communication with said light sources, wherein each light source corresponds with one of said containers when in position;   a first optical path means for guiding light from said light source to said corresponding container;   an optical signal sensing means in optical communication with the sample in said positioned containers; and   a second optical path means for guiding emitted light from the sample to said optical signal sensing means.   
     
     
         2 . The fluorometer of  claim 1 , further comprising:
 an excitation filter in said first optical path means for allowing transmission therethrough of an excitation wavelength in the light generated from each light source; and   an emission filter in said second optical path means for allowing transmission therethrough of emitted light from the sample and for substantially blocking transmission of wavelengths other than the wavelengths of said emitted light.   
     
     
         3 . The fluorometer of  claim 1  wherein said low heat-generating light sources are light emitting diodes. 
     
     
         4 . The fluorometer of  claim 3  wherein said light emitting diodes are blue and the potentially fluorescing sample includes a reagent capable of excitation by blue light. 
     
     
         5 . The fluorometer of  claim 3  wherein said containers comprise sample tubes. 
     
     
         6 . The fluorometer of  claim 5  wherein the positioning means is a sample tube holder or a multiplate. 
     
     
         7 . The fluorometer of  claim 6  wherein the sample tubes or the wells of the multiplate form an 8 by 12 array, and the light sources are arranged in a corresponding array. 
     
     
         8 . The fluorometer of  claim 3  further comprising means for uniformizing the output from the light emitting diodes. 
     
     
         9 . The fluorometer of  claim 8  wherein the uniformizing means comprises a silicon photodiode and associated circuitry. 
     
     
         10 . The fluorometer of  claim 8  wherein said uniformizing means comprises a calibration phosphor and a light emitting diode. 
     
     
         11 . The fluorometer of  claim 1  wherein said optical signal sensing means is selected from the group consisting of a photomultiplier tube, a charge coupled device type camera, a vacuum photodiode and an avalanche photodiode. 
     
     
         12 . The fluorometer of  claim 11  wherein said optical signal sensing means is a photomultiplier tube. 
     
     
         13 . The fluorometer of  claim 11  wherein said optical signal sensing means is a change coupled device type camera. 
     
     
         14 . The fluorometer of  claim 1  further comprising means for calibrating said optical signal sensing means. 
     
     
         15 . The fluorometer of  claim 14 , wherein said calibrating means comprises:
 (a) a light emitting diode emitting the approximate wavelength of an excited sample, and   (b) associated circuitry.   
     
     
         16 . The fluorometer of  claim 1  further comprising means for powering each of said low heat-generating light sources. 
     
     
         17 . The fluorometer of  claim 1  wherein said first optical path means comprises at least one lens. 
     
     
         18 . The fluorometer of  claim 17  wherein said lens is a fresnel lens. 
     
     
         19 . The fluorometer of  claim 18  wherein said fresnel lens is pixelized. 
     
     
         20 . The fluorometer of  claim 19  wherein said pixelized fresnel lens ranges in thickness from about 8 to about 15 mil. 
     
     
         21 . The fluorometer of  claim 16  wherein the first optical path means comprises a plurality of individual lenses, one such lens positioned proximate to each said light source. 
     
     
         22 . The fluorometer of  claim 16  wherein the first optical path means comprises a plurality of individual lens, one such lens positioned proximate to each positioned container. 
     
     
         23 . The fluorometer of  claim 1  wherein said first optical path means comprises at least one reflector. 
     
     
         24 . The fluorometer of  claim 2  wherein said emission filter is positioned proximate to said optical signal sensing means. 
     
     
         25 . The fluorometer of  claim 2  wherein said emission filter is sufficiently spaced apart from said optical signal sensing means as to confine emitted light from the fluorescing sample to an included conical angle of about 45°. 
     
     
         26 . The fluorometer of  claim 2  further comprising a plurality of optical filters, one such fiber for each container, through which emitted light from the fluorescing sample is transmitted through said emission filter to said optical signal sensing means. 
     
     
         27 . The fluorometer of  claim 1  further comprising programmable control means for controlling at least one function of the fluorometer. 
     
     
         28 . The fluorometer of  claim 1  further comprising means for measuring the fluorescence from each fluorescing sample, said measuring means being in electrical communication with said optical signal sensing means. 
     
     
         29 . A combined fluorometer and thermal cycler, comprising the fluorometer of  claim 1 , and a thermal cycler. 
     
     
         30 . The combined fluorometer and thermal cycler of  claim 29 , wherein the thermal cycler comprises:
 (a) a thermally controlled base holding in close contact a plurality of sample holders;   (b) a thermally controlled cover having a plurality of apertures corresponding to each sample holder, said cover in operative condition mechanically biasing each said sample holder into said close contact, each said aperture expanding outward from said cap; and   (c) programmable control means for controlling the temperature of said sample holders according to a selected protocol.   
     
     
         31 . The combined fluorometer and thermal cycler of  claim 30 , wherein the plurality of sample holders is a plurality of capped sample tubes or a multiplate. 
     
     
         32 . The combined fluorometer and thermal cycler of  claim 30  wherein said apertures expand outwardly toward the optical signal sensing means at an included angle of about 10 degrees. 
     
     
         33 . The combined fluorometer and thermal cycler of  claim 30  wherein said apertures are formed as a parabolic reflector. 
     
     
         34 . The combined fluorometer and thermal cycler of  claim 30  wherein said sample holder are integrally formed within the positioning means of the fluorometer. 
     
     
         35 . A method for analyzing polymerase chain reaction amplified material, comprising the steps of:
 positioning sample material subjected to polymerase chain reaction amplification into optical communication with the light sources of the fluorometer of  claim 1 ;   exposing the sample material to an excitation wavelength;   detecting the emitted light with the optical signal sensing means; and   comparing the emitted light level to a pre-determined reference level.   
     
     
         36 . The method of  claim 35  further comprising collecting melting curve data and identifying an unknown DNA according to algorithms predictive of an examined DNA sequence relative to teachings by known DNA sequences. 
     
     
         37 . The method of  claim 36  wherein the sample material was amplified by exposure to a selected DNA annealing/denaturing temperature range which comprises a single cycle. 
     
     
         38 . The method of  claim 36  wherein said sample material was amplified by exposure to a selected DNA annealing/denaturing temperature range which comprises multiple cycles. 
     
     
         39 . The method of  claim 35  wherein the emitted light detected has been subjected to means for uniformizing the output of the light sources in association with programmable means. 
     
     
         40 . The method of  claim 39 , wherein the means for uniformizing
 (a) comprises a silicon photodiode and its associated feed-back circuitry, and   (b) is located in a position where the silicon photodiode is reachable by the light from each of the light sources.   
     
     
         41 . A method for analyzing polymerase chain reaction amplified material, comprising the steps of:
 positioning sample material subjected to polymerase chain reaction amplification into optical communication with the light sources of the combined fluorometer and thermal cycler of  claim 29 ;   exposing the sample material to an excitation wavelength;   detecting the emitted light with the optical signal sensing means; and   comparing the emitted light level to a pre-determined reference level.