System and method for increasing the contrast of an image produced by an epifluorescence microscope

Abstract

The contrast of an image produced by epifluorescence microscopy may be increased by placing a dichroic reflector behind the sample. The dichroic reflector reflects the emission light emitted by the fluorescent tags in the sample back through the objective lens while allowing the shorter wavelength excitation light to pass through the sample holder.

1 . An epifluorescence microscope for imaging a biological sample having fluorescent tag molecules, the tag molecules emitting an emission light at an emission frequency when illuminated by an excitation light having an excitation frequency, the microscope comprising: 
 an excitation light source generating an excitation light;    a first dichroic mirror reflecting the excitation light;    an objective lens disposed to receive the excitation light reflected by the dichroic mirror and to illuminate the sample with the excitation light;    an imaging lens disposed to receive emission light from the sample through the objective lens and first dichroic mirror; and    a dichroic sample reflector disposed behind the sample reflecting the emission light back through the sample, objective lens, first dichroic mirror and imaging lens, while transmitting the excitation light through the reflector.    
   
   
       2 . The epifluorescence microscope of  claim 1  wherein the dichroic sample reflector is concave having a focal point disposed in the sample.  
   
   
       3 . The epifluorescence microscope of  claim 1  wherein said biological sample is placed directly on the dichroic sample reflector.  
   
   
       4 . A method for increasing the contrast of an image produced by an epifluorescence microscope of a sample emitting an emission light when illuminated by an excitation light comprising the steps of: 
 illuminating the sample with the excitation light;    collecting a first portion of the emission light;    reflecting a second portion of the emission light;    collecting the reflected portion of the emission light; and    producing an image using the collected first portion of the emission light and the collected reflected portion of the emission light.    
   
   
       5 . The method in  claim 4  wherein the reflected portion of the emission light is collected from a focal point disposed in said sample.  
   
   
       6 . The method of  claim 4  wherein said sample is placed directly on a dichroic sample reflector.