Quantified Fluorescence Microscopy

Abstract

The present invention relates to calibration apparatuses, methods or tools used in microscopy. A calibration tool for fluorescent microscopy includes a support, a solid surface layer including a fluorescent material, and a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of the surface layer. A first type of the calibration tool may include an adhesion promoter facilitating contact between the surface of the support and the solid surface layer including the fluorescent material, which is in contact with the opaque mask. A second type of the calibration tool may include the thin opaque mask fabricated (with or without an adhesion promoter) onto the support, and the solid surface layer including the fluorescent material located on the thin opaque mask.

1 . A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising: a polymer layer comprising at least one fluorescent agent, wherein said device has minimal local and global nonuniformities and is dimensioned for placement in an optical scanner.  
   
   
       2 . The device according to  claim 1 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       3 . The calibration device according to  claim 1 , wherein said polymer is a polyimide.  
   
   
       4 . The calibration device according to  claim 1 , wherein the thickness of said polymer layer ranges from about 2 microns to about 250 microns.  
   
   
       5 . The calibration device according to  claim 1 , wherein the thickness of said polymer layer ranges from about 1 micron to about 10 microns.  
   
   
       6 . The calibration device according to  claim 1 , wherein said at least one fluorescent agent absorbs and emits light in the portion of the electromagnetic spectrum to which a photomultiplyer tube of said optical scanner is sensitive.  
   
   
       7 . The calibration device according to  claim 1 , wherein said at least one fluorescent agent absorbs and emits light in the wavelength range selected from the group consisting of ultraviolet, visible and near infrared.  
   
   
       8 . The calibration device according to  claim 1 , wherein said polymer layer is a spin-coated polymer layer.  
   
   
       9 . A method for calibrating a biopolymeric array optical scanning system, said method comprising: (a) illuminating a surface of a calibration device with at least one light source, wherein said calibration device is a calibration device according to  claim 1;  (b) obtaining fluorescence data from said surface of said calibration device; and (c) calibrating said optical scanning system based upon said fluorescence data.  
   
   
       10 . The method according to  claim 9 , wherein said step of illuminating comprises illuminating said surface of said calibration device in the portion of the electromagnetic spectrum to which a photomultiplyer tube of said optical scanner is sensitive.  
   
   
       11 . The method according to  claim 9 , wherein said step of illuminating comprises illuminating said surface of said calibration device in the wavelength range selected from the group consisting of ultraviolet, visible and near infrared.  
   
   
       12 . The method according to  claim 9 , wherein said step of obtaining fluorescence data comprises detecting a signal related to the intensity of emitted light from said fluorescent agent.  
   
   
       13 . The method according to  claim 9 , wherein said fluorescent agent(s) is distributed substantially uniformly throughout said surface.  
   
   
       14 . A method for performing a hybridization assay, said method comprising: (a) calibrating an optical scanner with a calibration device, wherein said calibration device is calibration device according to  claim 1 , (b) performing a hybridization assay with at least one array, and (c) scanning said array with said calibrated optical scanner.  
   
   
       15 . A method for manufacturing a calibration device, said method comprising spin-coating a composition onto a substrate to produce a calibration device according to  claim 1 .  
   
   
       16 . A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising: a polymer layer comprising at least one fluorescent agent, wherein said device has minimal local and global nonuniformities; and a transparent substrate; wherein said device is dimensioned for placement in an optical scanner.  
   
   
       17 . The calibration device according to  claim 16 , wherein said transparent substrate is glass.  
   
   
       18 . The calibration device according to  claim 17 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       19 . The calibration device according to  claim 16 , wherein the thickness of said polymer layer ranges from about 1 micron to about 10 microns.  
   
   
       20 . A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising: a polymer layer comprising at least one fluorescent agent, wherein said device has minimal local and global nonuniformities; and a substrate; wherein said device is dimensioned for placement in an optical scanner.  
   
   
       21 . The calibration device according to  claim 20 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       22 . The calibration device according to  claim 20 , wherein the thickness of said polymer layer ranges from about 1 micron to about 10 microns.  
   
   
       23 . An optical scanner comprising a calibration device according to  claim 1 .  
   
   
       24 . A calibration device for confirming or calibrating a biopolymeric array optical scanner, said device comprising: a polymer layer comprising at least one fluorescent agent, wherein said device is dimensioned for placement in an optical scanner, and wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       25 . The device according to  claim 24 , wherein said polymer layer is of uniform thickness.  
   
   
       26 . A method for manufacturing a calibration tool for a fluorescent microscope that is useful to image an array, comprising 
 providing a support, providing a solid surface layer including a fluorescent material, and    fabricating a thin opaque mask of non-fluorescent material defining reference feature openings having selected dimensions exposing portions of the surface layer; forming the solid surface layer by depositing it onto the support, comprising depositing an adhesion promoting layer onto the support for forming the solid surface layer including the fluorescent material, the deposition includes evaporating or sputtering, the depositing includes spin coating to form the solid surface layer including the fluorescent material.    
   
   
       27 . A method for using a fluorescent microscope to image a bioploymer array, comprising exciting the array at 473 nanometers, producing ½ volt at a photomultiplyer tube in the microscope, using a fluorophore having a fluorescent efficiency that is approximately 1×10 −6  or higher, and using a calibration tool.