Scanning spectrophotometer for high throughput fluorescence detection

Abstract

A fluorescence spectrophotometer having an excitation double monochromator, a coaxial excitation/emission light transfer module, and an emission double monochromator. Each monochromator includes a pair of holographic concave gratings mounted to precisely select a desired band of wavelengths from incoming broadband light without using other optical elements, such as mirrors. Selected excitation light is directed into a sample well by a light transfer module that includes a coaxial excitation mirror positioned to direct excitation light directly to the bottom of a well of a multi-well plate. Fluorescence emission light that exits the well opening is collected by a relatively large coaxial emission mirror. The collected emission light is wavelength selected by the emission double monochromator. Selected emission light is detected by a photodetector module.

1 . A double monochromator including:
 (a) an entrance slit for accepting light;   (b) a first optical grating positioned to intercept and disperse the accepted light from the entrance slit;   (c) a first selection slit positioned to intercept a least part of the dispersed light from the first optical grating and select and pass a narrowed range of wavelengths from such dispersed light;   (d) a second optical grating positioned to intercept and disperse the passed light from the first selection slit; and   (e) a second selection slit positioned to intercept at least part of the dispersed light from the second optical grating and select and pass a narrowed range of wavelengths from such dispersed light.   
   
   
       2 . The double monochromator of  claim 1 , wherein the first optical grating and the second optical grating are both concave gratings. 
   
   
       3 . The double monochromator of  claim 2 , wherein the concave gratings are holographic concave gratings. 
   
   
       4 . The double monochromator of  claim 1 , wherein the first optical grating and the second optical grating pivot about axes of rotation for selecting a desired range of wavelengths of light as a function of angle of rotation. 
   
   
       5 . The double monochromator of  claim 1 , further including a band drive, coupled to each of the first optical grating and the second optical grating, for rotating the first optical grating and the second optical grating synchronously. 
   
   
       6 . The double monochromator of  claim 1 , wherein the double monochromator is an emission double monochromator and is positioned between a sample and a photodetector, the sample emitting light responsive to illumination by an excitation light. 
   
   
       7 . The double monochromator of  claim 1 , wherein the double monochromator is an excitation double monochromator positioned between a light source and sample. 
   
   
       8 . The double monochromator of  claim 6 , wherein the accepted light is emission light. 
   
   
       9 . The double monochromator of  claim 7 , wherein the accepted light is excitation light. 
   
   
       10 . The excitation double monochromator of  claim 7 , wherein the second selection slit is a pinhole slit. 
   
   
       11 . The excitation double monochromator of  claim 10 , wherein the pinhole slit directs light onto a specimen and wherein the imaged light can be used to excite fluorescence. 
   
   
       12 . The excitation double monochromator of  claim 9 , further comprising:
 a light source;   a multi-well plate wherein at least one of the light source and the double monochromator is translatable from a first position to a second position, the second position being located below the multi-well plate;   wherein one or more wells of the multi-well plate have a transparent bottom substrate; and,   an excitation mirror positioned to direct the excitation light through the transparent bottom substrate.   
   
   
       13 . The excitation double monochromator of  claim 12 , further comprising an emission mirror operative to collect emitted light, the emitted light being from the well. 
   
   
       14 . The excitation monochromator of  claim 9 , further comprising a set of one or more redirection mirrors operative to receive the excitation light. 
   
   
       15 . The excitation monochromator of  claim 14  further comprising:
 a well having a transparent bottom substrate; and,   an excitation mirror operative to direct the excitation light through the transparent bottom substrate of the well.   
   
   
       16 . The double monochromator of  claim 3 , wherein the holographic concave gratings are operatively mounted to select a desired band of wavelengths from the accepted light. 
   
   
       17 . The double monochromator of  claim 1 , wherein the accepted light is polarized; the double monochromator further comprising:
 a first polarizing filter operative to transmit light in a plane which is parallel to the plane of the polarized accepted light;   a second polarizing filter operative to transmit light in any plane which is not parallel to the plane of the polarized accepted light; and,   a polarizing filter holder selectively operative to insert one of the first polarizing filter or the second polarizing filter into the path of the accepted light.