Use of Microparticle Additives to Simultaneously Enable Artifact-Free Image Registration, Auto-Focusing, and Chromatic Aberration Correction in Microscopy

Abstract

High-contrast, high-density cell-sized microparticles are introduced into a cell-containing solution prior to the solution being spread onto a planar substrate for imaging. The microparticles facilitate both the process of imager autofocusing and the subsequent registration of multiple images taken of regions of the substrate. The microparticles can further facilitate the correction of chromatic aberration.

What is claimed is: 
     
         1 . A method for using an imager having an auto-focuser to acquire target information from subject targets on a planar substrate, comprising the steps of:
 forming a mixture of subject targets and facilitative targets on a planar substrate;   in a first focusing step, using the auto-focuser, focusing the imager with respect to facilitative targets within a first region; and   in a first imaging step, without refocusing the imager, using the imager to acquire information within the first region from at least subject targets.   
     
     
         2 . The method of  claim 1 , wherein the subject targets are cells and the facilitative targets are microparticles. 
     
     
         3 . The method of  claim 1 , wherein during at least one of the first focusing step and the first imaging step the imager acquires first region facilitative target information,
 in a second focusing step, using the auto-focuser, focusing the imager with respect to facilitative targets within a second region;   in a second imaging step, without refocusing the imager, using the imager to acquire target information within the second region from at least subject targets, wherein during at least one of the second focusing step and the second imaging step the imager acquires second region facilitative target information; and   registering the acquired target information from the subject targets within the first and second regions, using the first region facilitative target information and the second region facilitative target information.   
     
     
         4 . The method of  claim 3 , wherein the subject targets are cells and the facilitative targets are microparticles. 
     
     
         5 . The method of  claim 3 , wherein the registration step is performed using first region facilitative target information and second region facilitative target information obtained, respectively, during the first and the second focusing steps. 
     
     
         6 . The method of  claim 1 , wherein the first focusing and first imaging steps are performed at a first wavelength band, and further comprising the steps of:
 in a second focusing step, using the auto-focuser, focusing the imager at a second wavelength band with respect to facilitative targets within the first region; and   in a second imaging step, without refocusing the imager, using the imager to acquire information at the second wavelength band from subject targets within the first region.   
     
     
         7 . The method of  claim 6 , further comprising the step of overlaying the information acquired from the subject targets at the first and the second wavelength bands. 
     
     
         8 . A method for registering target information acquired by imaging subject targets within a plurality of regions in an examination zone of a planar substrate, comprising the steps of:
 forming a mixture of subject targets and facilitative targets on a planar substrate;   using the imager, acquiring target information from subject targets and facilitative targets within a first region within the examination zone;   using the imager, acquiring target information from subject targets and facilitative targets within a second region within the examination zone; and   registering the acquired target information from the subject targets within the first and the second regions, using the information acquired from the facilitative targets within the first and second regions.   
     
     
         9 . The method of  claim 8 , wherein the subject targets are cells and the facilitative targets are microparticles. 
     
     
         10 . A method for correcting chromatic aberration in an optical image of subject targets, comprising the steps of
 forming a mixture of subject targets and facilitative targets on a planar substrate;   acquiring first wavelength image data and second wavelength image data for the mixture of subject targets and facilitative targets on the planar substrate, the first wavelength image data and the second wavelength image data comprising, for at least one facilitative target, first wavelength apparent position data and second wavelength apparent position data;   generating a transformation function from the difference between the first wavelength apparent position and the second wavelength apparent position for the at least one facilitative target; and   applying the transformation function to at least one subject target to correct for chromatic aberration.   
     
     
         11 . The method of  claim 10 , wherein the subject targets are cells and the facilitative targets are microparticles. 
     
     
         12 . The method of  claim 10 , wherein the first and the second wavelength apparent position data include representations of positions along an axis orthogonal to the planar substrate. 
     
     
         13 . The method of  claim 10 , wherein the first and the second wavelength apparent position data include representations of positions in a plane coplanar with the planar substrate. 
     
     
         14 . The method of  claim 13 , wherein the first and the second wavelength apparent position data further include representations of positions along an axis orthogonal to the planar substrate. 
     
     
         15 . A method for using an imager having an auto-focuser to acquire target information from subject targets on a planar substrate, comprising the steps of:
 forming a mixture of subject targets and facilitative targets on a planar substrate;   using the auto-focuser, focusing the imager with respect to facilitative targets within a first region to obtain at least one optimized focal parameter;   refining the at least one optimized focal parameter to determine a refined focal parameter for subject targets;   refocusing the imager using the refined focal parameter; and   acquiring information from subject targets within the first region.   
     
     
         16 . The method of  claim 15 , wherein the subject targets are cells and the facilitative targets are microparticles. 
     
     
         17 . The method of  claim 15 , wherein the refining consists of adjusting the at least one optimized focal parameter by a fixed offset. 
     
     
         18 . The method of  claim 16 , wherein the subject targets are cells and the facilitative targets are microparticles.