Systems and methods for detection of labeled materials

Abstract

Labeled targets on a support synthesized with polymer sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, can be detected by exposing marked regions of sample to radiation from a source and detecting the emission therefrom, and repeating the steps of exposition and detection until the sample is completely examined.

What is claimed is:  
     
         1 . A system for detecting marked regions on a surface of a substrate, the system comprising: 
 an excitation radiation source;    a focusing system for focusing radiation from said excitation radiation source onto a selected region of said surface of said substrate, said focusing system including an objective lens having a ratio of scanning field diameter to focused spot diameter of greater than about 2000, and a numerical aperture greater than about 0.2;    a radiation direction system for scanning said focused excitation radiation across said surface of said substrate at a rate of at least 5 image lines/second;    a detector for detecting an emission from said surface of said substrate in response to said excitation is radiation, wherein said objective lens receives said emission and transmits the emission to the detector; and    a data acquisition system for recording an amount of said emission detected as a function of a position on said surface of said substrate from which said emission was emitted.    
     
     
         2 . The system of    claim 1   , wherein said focusing system has a ratio of scanning field diameter to focused spot diameter of greater than 3000.  
     
     
         3 . The system of    claim 1   , wherein said focusing system has a ratio of scanning field diameter to focused spot diameter of greater than 4000.  
     
     
         4 . The system of    claim 1   , wherein said focusing system focuses said excitation radiation in a spot having a diameter of less than about 10 μm.  
     
     
         5 . The system of    claim 1   , wherein said focusing system focuses said excitation radiation on said surface of said substrate in a spot having a diameter of less than about 5 μm.  
     
     
         6 . The system of    claim 1    wherein said focusing system focuses said excitation radiation on said surface of said substrate in a spot having a diameter of about 3 μm.  
     
     
         7 . The system of    claim 1   , wherein said scanning field diameter is greater than about 10 mm.  
     
     
         8 . The system of    claim 1   , wherein said scanning field diameter is about 14 mm.  
     
     
         9 . The system of    claim 1   , wherein said numerical aperture is greater than about 0.25.  
     
     
         10 . The system of    claim 1   , wherein said focusing system is achromatic.  
     
     
         11 . The system of    claim 1   , wherein said radiation direction system is capable of scanning a spot across the substrate at a rate of at least 10 image lines/second.  
     
     
         12 . The system of    claim 1   , wherein said radiation direction system is capable of scanning a spot across the substrate at a rate of at least 30 image lines/second.  
     
     
         13 . The system of    claim 1   , wherein said radiation direction system comprises an angularly oscillating mirror or a rotating polyhedral mirror.  
     
     
         14 . The system of    claim 1   , further comprising a translation stage upon which said substrate is mounted, said translation stage being moveable in at least one dimension perpendicular to an optical axis of said objective lens.  
     
     
         15 . The system of    claim 1   , further comprising an autofocus system for placing said surface of said substrate in a focal plane of said focusing system.  
     
     
         16 . The system of    claim 1   , further comprising a translation stage upon which said substrate is mounted, said translation stage being moveable in at least one dimension parallel to an optical axis of said focusing system and at least one dimension perpendicular to said optical axis of said focusing system.  
     
     
         17 . The system of    claim 1    further comprising a collection system comprising: 
 collection optics for collecting fluorescence emitted from said surface of said substrate and for collecting an excitation radiation reflected from said surface of said substrate;  
 separation optics for separating said emitted fluorescence from an excitation radiation reflected from said surface of said substrate and focusing said fluorescence through a confocal pinhole; and  
 a recorder responsive to said fluorescence, for recording an amount of said fluorescence focused through said confocal pinhole.  
 
     
     
         18 . The system of    claim 1   , wherein said surface of said substrate comprises a plurality of distinct polymer sequences in different known locations on said surface.  
     
     
         19 . The system of    claim 18   , wherein each of said different polymer sequences is contained in a feature having at least one of a width or length dimension of less than about 50 μm.  
     
     
         20 . The system of    claim 1    further comprising a processor for processing and storing said signal so as to generate a 2-dimensional image of said sample.  
     
     
         21 . The system of    claim 1    further comprising a body for immobilizing said substrate, the substrate having at least a first surface with a sample thereon, the body comprising: 
 a mounting surface;  
 a cavity in said mounting surface, said first surface mated to said mounting surface for sealing said cavity, said sample being in fluid communication with said cavity, said cavity having a bottom surface comprising a light absorptive material;  
 an inlet and an outlet being in communication with said cavity such that fluid flowing into said cavity for contacting said sample flows through said inlet and fluid flowing out of said cavity flows through said outlet; and  
 a temperature controller for controlling the temperature in said cavity.  
 
     
     
         22 . The system of    claim 21    wherein said temperature controller comprises a thermoelectric cooler.  
     
     
         23 . A system for detecting marked regions on a surface of a substrate, the system comprising: 
 an excitation radiation source;    first focusing optics for focusing said excitation radiation on said surface of said substrate in a spot having a diameter no greater than 5 μm, said focusing optics including an objective lens;    a reciprocating radiation direction system for scanning said spot linearly across said surface of said substrate, said spot having a travel distance of at least 10 mm;    an optical train for separating emissions from said surface of said substrate from excitation radiation reflected from said surface of said substrate, said optical train including said objective lens; and    an autofocus system for automatically placing said surface of said substrate in a focal plane of said first focusing optics.    
     
     
         24 . The system of    claim 23   , wherein said optical train includes a dichroic beam splitter for separating fluorescence emitted from said surface of said substrate from excitation radiation reflected from said surface of said substrate.  
     
     
         25 . The system of    claim 23   , wherein said autofocus system comprises: 
 second focusing optics for focusing said excitation radiation reflected from said surface of said substrate through a confocal pinhole onto a photodiode; and    a translation stage upon which a substrate is mounted, said stage being capable of moving in a direction parallel with an optical axis of said first focusing optics for moving said substrate into a focal plane of said first focusing optics.    
     
     
         26 . The system of    claim 23    further comprising a detector for detecting an emission from said surface of said substrate in response to said excitation radiation, and a data acquisition system for recording an amount of said emission detected as a function of a position on said surface of said substrate from which said emission was emitted.  
     
     
         27 . A method of scanning a polymer array having a plurality of different polymer sequences, each of said different polymer sequences being immobilized on a surface of a substrate in a different known location, to identify which polymer sequence on said array is bound by a target molecule, the method comprising: 
 focusing an excitation radiation source upon said surface of said substrate with an objective lens;    scanning said excitation radiation across said surface of said substrate at a speed of at least about 5 image lines/second;    collecting emissions from said surface of said substrate in response to said excitation radiation with said objection lens;    recording said emissions as a function of a position on said surface of said substrate, said position indicating the polymer sequence on said array is bound by a target molecule.    
     
     
         28 . The method of    claim 27   , wherein said polymer array comprises a plurality of different oligonucleotide sequences immobilized on said surface of said substrate in different known locations.  
     
     
         29 . The method of    claim 27    further comprising focusing the excitation radiation source to a spot having a diameter of about 3 μm and scanning the spot in linear direction across the substrate for at least 14 mm.  
     
     
         30 . The method of    claim 27    further comprising the steps of: 
 immobilizing said substrate on a body;  
 exciting said sample on said substrate with an excitation radiation having a first wavelength from an electromagnetic radiation source, said excitation radiation  
 detecting a response radiation having a second wavelength in response to said excitation radiation, said response radiation representing an image of said plurality of regions;  
 exciting a subsequent plurality of regions on said sample;  
 processing and storing said response radiation to generate a 2-dimensional image of said sample; and  
 auto-focusing said sample in a focal plane of said excitation radiation.  
 
     
     
         31 . The method as recited in    claim 30    wherein said body comprises a mounting surface having a cavity thereon, said substrate being immobilized on said mounting surface such that said sample is in fluid communication with said reaction chamber, said reaction chamber comprising a inlet and a outlet for flowing fluids into and through said reaction chamber.  
     
     
         32 . The method as recited in    claim 31    wherein said body further comprises a temperature controller for controlling the temperature in said cavity.  
     
     
         33 . The method as recited in    claim 27    wherein said excitation radiation is scanned across said surface of said substrate at a speed of at least about 10 image lines/second.  
     
     
         34 . The method as recited in    claim 27    wherein said excitation radiation is scanned across said surface of said substrate at a speed of at least about 30 image lines/second.