Rare cell detection using flat-panel imager and chemiluminescent or radioisotopic tags

Abstract

Disclosed is a method using a large area flat panel imager which is specifically adapted for rare cell detection methods. The method generally includes an imager having a sample receiving surface which can provide a digital or electronic image of a sample deposited on the surface. The method also includes a selectively positionable microscope and/or camera which are used to obtain high resolution images of the deposited samples. An electronic controller can also be used in conjunction with the imager, microscope, and/or camera to selectively position at least one of those components to focus on desired regions of the deposited sample. The noted method is particularly adapted for use with chemiluminescence or other tagging technologies.

1 . A method for detecting the presence of tagged biological agents in a sample, the method comprising:
 providing a system having an imager, a microscope, a camera, and a sample receiving surface;   providing a sample containing a biological agent to be detected;   depositing the sample upon the sample receiving surface of the system;   tagging the biological agent to be detected within the sample;   obtaining a digital image of the deposited tagged sample from the system;   analyzing the digital image to select one or more regions of the tagged sample for high-resolution examination;   positioning the imager, microscope, and camera relative to each other so the microscope can image a selected region of the tagged sample determined in the analyzing step;   obtaining a second digital image of the selected region of the tagged sample; and   confirming the detection of the tagged biological agent.   
   
   
       2 . The method of  claim 1 , wherein the step of tagging is performed by tagging the biological agent using an antibody-based method or a DNA-based method with linked-enzyme(s) for chemiluminescent detection and/or with fluorochrome(s) or radioisotope(s). 
   
   
       3 . The method of  claim 1 , wherein multiple tagging methods are performed on the same sample. 
   
   
       4 . The method of  claim 1 , wherein the step of depositing is performed by depositing the sample on a substrate and then placing the substrate on the sample receiving surface of the system. 
   
   
       5 . The method of  claim 1 , wherein the step of obtaining the first digital image from the system is performed by detecting a range of wavelengths emitted by the tagged sample. 
   
   
       6 . The method of  claim 1 , wherein the step of analyzing is performed by determining the regions of the sample which emitted light at a range of wavelengths above a threshold, the threshold being determined by human or electronic means. 
   
   
       7 . The method of  claim 1  wherein the step of positioning is performed using electronic positioning equipment to move one or more of the imager, the microscope, and the camera. 
   
   
       8 . The method of  claim 1 , wherein the step of obtaining the second digital image is performed by using the microscope in conjunction with the camera to capture the second digital image. 
   
   
       9 . The method of  claim 1  wherein the second digital image has a higher resolution than the first digital image. 
   
   
       10 . A method for detecting the presence of tagged biological agents in a sample by use of a system having, (i) a large area imager having an image forming surface, the imager being able to form digital or electronic images of the sample free from intermediate image forming lenses, (i)) a selectively positionable microscope and camera in viewing relation with the image forming surface, the camera adapted to acquire an image through the microscope in the form of digital data, and (iii) an electronic controller in communication with the imager, microscope, and camera, and configured to position at least one of the imager, microscope, and camera relative to each other in order to image selected regions of the image forming surface, the method comprising:
 providing a sample containing a biological agent to be detected;   depositing the sample upon the sample receiving surface of the system;   tagging the biological agent to be detected within the sample;   obtaining a digital image of the deposited tagged sample from the system;   analyzing the digital image to select one or more regions of the tagged sample for high-resolution examination;   positioning the imager, microscope, and camera relative to each other so the microscope can image a selected region of the tagged sample determined in the analyzing step;   obtaining a second digital image of the selected region of the tagged sample; and   confirming the detection of the tagged biological agent.   
   
   
       11 . The system of  claim 10 , wherein the microscope contains an objective lens of magnification power of from about 10 to about 100. 
   
   
       12 . The method of  claim 10 , wherein the system further comprises one or more filters, the filters substantially passing light within a range of wavelengths and substantially blocking light outside the range of wavelengths. 
   
   
       13 . The method of  claim 10  wherein the sample receiving surface includes a coating that facilitates binding of cells thereto. 
   
   
       14 . The method of  claim 13  wherein the coating is compatible with chemiluminescent detection. 
   
   
       15 . The method of  claim 10 , further including matching a wavelength of a maximum quantum efficiency of the large area imager to a wavelength of an emitted photon from a chemiluminescent reagent in the sample. 
   
   
       16 . The method of  claim 10 , further including designing a quantum efficiency of the large area imager to match a wavelength of photons emitted from a selected reagent in the sample which are emitted at a rate of between about 400 to about 700 nm. 
   
   
       17 . The method of  claim 10 , further including adding noise of individual frames together such that the total noise can be expressed by a calculation:
   σ total =√{square root over ([(1069500)+[(4.5×10 −15   ×t )/(1.6×10 −19   ×n )]]× n )}{square root over ([(1069500)+[(4.5×10 −15   ×t )/(1.6×10 −19   ×n )]]× n )}{square root over ([(1069500)+[(4.5×10 −15   ×t )/(1.6×10 −19   ×n )]]× n )}   
     where t is the total integration time and n is the number of readouts during the entire exposure process 
   
   
       18 . The method of  claim 17 , wherein as a frequency of the system increases, a number of photons emitted increases compared to the photons used in the calculation, resulting in shorter integration times. 
   
   
       19 . The method of  claim 18 , wherein the number of photons emitted is tenfold higher than used in the calculation, resulting in tenfold shorter integration times. 
   
   
       20 . A method for detecting the presence of tagged biological agents in a sample, the method comprising:
 forming on a large area imager having an image forming surface, digital or electronic images of the sample free from intermediate image forming lenses;   positioning a selectively positionable microscope and camera in viewing relation with the image forming surface;   acquiring by the camera an image through the microscope in the form of digital data;   positioning at least one of the imager, microscope, and camera relative to each other in order to image selected regions of the image forming surface and integrate a time period (t), and a number (n) of read-out frames with a frame time (t/n); and   forming images by integrating the images over the integrating time period (t), including breaking the image integrating time period (t) into the number (n) of read-out frames to avoid noise saturation of the images.