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 thin 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 tool for fluorescent microscopy comprising a support on which is carried a solid surface layer comprised of effective fluorophores, and a thin mask of non-fluorescent material defining reference feature openings of limited dimensions exposing portions of the fluorophore-comprising surface layer.  
   
   
       2 - 44 . (canceled)  
   
   
       45 . 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.    
   
   
       46 . The device according to  claim 45 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       47 . The calibration device according to  claim 45 , wherein said polymer is selected from the group consisting of acrylates, epoxides, urethanes, polycarbonates, polyolefins, polyetherketones, polyesters, polystyrenes, polyethylstyrene, polysiloxanes, and copolymers thereof.  
   
   
       48 . The calibration device according to  claim 45 , wherein said polymer is polymethyl-methacrylate.  
   
   
       49 . The calibration device according to  claim 45 , wherein the thickness of said polymer layer ranges from about 0.25 micron to about 10 microns.  
   
   
       50 . The calibration device according to  claim 45 , wherein the thickness of said polymer layer ranges from about 0.4 micron to about 1 micron.  
   
   
       51 . The calibration device according to  claim 45 , wherein said device comprises a single polymer layer.  
   
   
       52 . The calibration device according to  claim 45 , wherein said device comprises a plurality of polymer layers.  
   
   
       53 . The calibration device according to  claim 45 , wherein said at least one fluorescent agent is present in said polymer in a final concentration ranging from about 1 ppm to about 5000 ppm.  
   
   
       54 . The calibration device according to  claim 45 , wherein said at least one fluorescent agent absorbs and emits light in the portion of the electromagnetic spectrum to which a photomultiplier tube of said optical scanner is sensitive.  
   
   
       55 . The calibration device according to  claim 45 , wherein said at least one fluorescent agent absorbs and emits light in the wavelength range selected from the group consisting of ultraviolet, visible and infrared.  
   
   
       56 . The calibration device according to  claim 45 , wherein said global nonuniformity of said calibration device is less than about 5%.  
   
   
       57 . The calibration device according to  claim 45 , wherein said local nonuniformity of said calibration device is less than about 5%.  
   
   
       58 . The calibration device according to  claim 45 , wherein said polymer layer comprises at least two fluorescent agents.  
   
   
       59 . The calibration device according to  claim 45 , wherein said polymer layer is selected from the group consisting of a spin-coated polymer layer, a draw coated polymer layer, a roller coated polymer layer, an electrodeposited polymer layer and a sprayed polymer layer.  
   
   
       60 . 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 45;     (b) obtaining fluorescence data from said surface of said calibration device; and    (c) calibrating said optical scanning system based upon said fluorescence data.    
   
   
       61 . The method according to  claim 60 , wherein said step of illuminating comprises illuminating said surface of said calibration device in the portion of the electromagnetic spectrum to which a photomultiplier tube of said optical scanner is sensitive.  
   
   
       62 . The method according to  claim 60 , 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 infrared.  
   
   
       63 . The method according to  claim 60 , wherein said step of obtaining fluorescence data comprises detecting a signal related to the intensity of emitted light from said fluorescent agent.  
   
   
       64 . The method according to  claim 60 , wherein said step of calibrating comprises calibrating the scale factor of said system.  
   
   
       65 . The method of  claim 64 , wherein said scale factor calibration comprises adjusting the sensitivity of an optical detector of said system.  
   
   
       66 . The method according to  claim 60 , wherein said step of calibrating comprises calibrating the focus position of said system.  
   
   
       67 . The method according to  claim 66 , wherein said focus position calibration comprises adjusting the distance between a scanning stage and a lens of said system.  
   
   
       68 . The method according to  claim 60 , wherein said step of calibrating comprises calibrating the dynamic focus of said system.  
   
   
       69 . The method according to  claim 68 , wherein said dynamic focus calibration comprises adjusting the rate of speed at which an optical stage of said system travels.  
   
   
       70 . The method according to  claim 60 , wherein said step of calibrating comprises determining the amount of oscillation in an intensity image and adjusting the rate of speed of said optical stage according to said oscillation data.  
   
   
       71 . The method according to  claim 60 , wherein said step of calibrating comprises calibrating at least one scanner mirror of said system.  
   
   
       72 . The method according to  claim 71 , wherein said at least one scanner mirror calibration comprises adjusting said at least one scanner mirror to synchronize the light beams of said system.  
   
   
       73 . The method according to  claim 60 , further comprising the steps of subtracting a background signal from said obtained fluorescent data to obtain a background corrected value.  
   
   
       74 . The method according to  claim 60 , wherein said fluorescent agent(s) is distributed substantially uniformly throughout said surface.  
   
   
       75 . The method according to  claim 60 , further comprising the step of verifying the jitter of said optical scanning system.  
   
   
       76 . 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 45 ,    (b) performing a hybridization assay with at least one array, and    (c) scanning said array with said calibrated optical scanner.    
   
   
       77 . A method comprising forwarding data representing a result of a scan obtained by the method of  claim 76 .  
   
   
       78 . The method according to  claim 77 , wherein said data is transmitted to a remote location.  
   
   
       79 . A method comprising receiving data representing a result of an interrogation obtained by the method of  claim 77 .  
   
   
       80 . 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 45 .  
   
   
       81 . The method according to  claim 77 , further comprising photobleaching at least one region of said device.  
   
   
       82 . A kit for calibrating a biopolymeric array optical scanner, said kit comprising: 
 (a) at least one device according to  claim 45;  and    (b) a substrate comprising instruction for using said device to calibrate a biopolymeric array optical scanner.    
   
   
       83 . A kit for calibrating a biopolymeric array optical scanner, said kit comprising: 
 (a) at least one device according to  claim 45;  and    (b) an array.    
   
   
       84 . The device according to  claim 45 , wherein said polymer layer is present on a substrate having a length ranging from about 4 mm to 200 mm.  
   
   
       85 . The device according to  claim 84 , wherein said substrate has a length ranging from about 4 mm to 150 mm.  
   
   
       86 . The device according to  claim 85 , wherein said substrate has a length ranging from about 4 mm to 125 mm.  
   
   
       87 . The device according to  claim 45 , wherein said polymer layer is present on a substrate having a width ranging from about 4 mm to 200 mm.  
   
   
       88 . The device according to  claim 87 , wherein said substrate has a width ranging from about 4 mm to 120 mm.  
   
   
       89 . The device according to  claim 88 , wherein said substrate has a width ranging from about 4 mm to 80 mm.  
   
   
       90 . The device according to  claim 45 , wherein said polymer layer is present on a substrate having a thickness ranging from about 0.01 mm to 5.0 mm.  
   
   
       91 . The device according to  claim 90 , wherein said substrate has a thickness ranging from about 0.1 mm to 2 mm.  
   
   
       92 . The device according to  claim 90 , wherein said subsrate has a thickness ranging from about 0.2 mm to 1 mm.  
   
   
       93 . 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.    
   
   
       94 . The calibration device according to  claim 93 , wherein said transparent substrate is glass.  
   
   
       95 . The calibration device according to  claim 94 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       96 . The calibration device according to  claim 93 , wherein the thickness of said polymer layer ranges from about 0.25 micron to about 10 microns.  
   
   
       97 . The calibration device according to  claim 93 , wherein said global nonuniformity of said calibration device is less than about 5%.  
   
   
       98 . The calibration device according to  claim 93 , wherein said local nonuniformity of said calibration device is less than about 5%.  
   
   
       99 . 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 having a length ranging from about 4 mm to 200 mm, a width ranging from about 4 mm to 200 mm and a thickness ranging from about 0.01 mm to 5.0 mm;    wherein said device is dimensioned for placement in an optical scanner.    
   
   
       100 . The calibration device according to  claim 99 , wherein said substrate is transparent.  
   
   
       101 . The calibration device according to  claim 99 , wherein said at least one fluorescent agent is distributed substantially uniformly throughout said polymer.  
   
   
       102 . The calibration device according to  claim 99 , wherein the thickness of said polymer layer ranges from about 0.25 micron to about 10 microns.  
   
   
       103 . The calibration device according to  claim 99 , wherein said global nonuniformity of said calibration device is less than about 5%.  
   
   
       104 . The calibration device according to  claim 99 , wherein said local nonuniformity of said calibration device is less than about 5%.  
   
   
       105 . An optical scanner comprising a calibration device according to  claim 45.