Ultrahigh resolution multi-color colocalization of single fluorescent probes

Abstract

A novel optical ruler based on ultrahigh-resolution colocalization of single fluorescent probes is described. Two unique families of fluorophores are used, namely energy-transfer fluorescent beads and semiconductor nanocrystal (NC) quantum dots, that can be excited by a single laser wavelength but emit at different wavelengths. A novel multicolor sample-scanning confocal microscope was constructed which allows one to image each fluorescent light emitter, free of chromatic aberrations, by scanning the sample with nanometer scale steps using a piezo-scanner. The resulting spots are accurately localized by fitting them to the known shape of the excitation point-spread-function of the microscope.

1 - 20 . (canceled)  
     
     
         21 . A apparatus for the colocalization of two or more species of interest in a sample comprising: 
 a scanner capable of supporting a sample and moving the sample;    a single wavelength light source capable of exciting the sample;    a device capable of separating the respective distinctive emission characteristics of the two or more species of interest; and    a device capable of detecting the emitted light from the two or more species of interest.    
     
     
         22 . The apparatus of  claim 21 , where the device capable of detecting the emitted light from the two or more species of interest detects emitted light simultaneously.  
     
     
         23 . The apparatus of  claim 21 , where the light source is a laser.  
     
     
         24 . The apparatus of  claim 23 , where the light source is a laser with wavelengths and power capable of exciting single photon processes.  
     
     
         25 . The apparatus of  claim 23 , where the light source is a laser with wavelengths and power capable of exciting multiple photon processes.  
     
     
         26 . The apparatus of  claim 21 , where the scanner is capable of moving a predetermined distance of from about  1  angstrom up to a distance of about 10 microns.  
     
     
         27 . The apparatus of  claim 21 , where the scanner is capble of moving a predetermined distance of about 25 nm.  
     
     
         28 . The apparatus of  claim 21 , where the device capable of detecting emitted light is a photodiode or a charge coupled device.  
     
     
         29 . The apparatus of  claim 23 , where the laser is a pulsed laser.  
     
     
         30 - 31 . (canceled)  
     
     
         32 . The apparatus of  claim 21 , where the scanner is a closed-loop, 3 axis, piezo-scanner.  
     
     
         33 . The apparatus of  claim 21 , where the scanner is an open-loop scanner.  
     
     
         34 . The apparatus of  claim 21 , where the scanner performs a two dimensional scan.  
     
     
         35 . The apparatus of  claim 21 , where the scanner performs a three dimensional scan.  
     
     
         36 . The apparatus of  claim 21 , wherein there is included a confocal pinhole.  
     
     
         37 - 40 . (canceled)  
     
     
         41 . The apparatus of  claim 21 , where the device capable of detecting the emitted light is a point detector.