Wavelength multiplexed quantitative differential interference contrast microscopy

Abstract

A differential interference contrast (DIC) microscope system is provided comprising: (a) an illumination source for illuminating a sample ; (b) a lens system for viewing the illuminated sample, including an objective, defining an optical axis; (c) at least one detector system for receiving a sample image; (d) mechanisms for wavelength multiplexing the shear direction or shear magnitude or both on the sample and demultiplexing the resultant DIC images on the detector; and (e) a mechanism for modulating the phase of the interference image. Various approaches are disclosed to accomplish wavelength multiplexing of shear direction and demultiplexing the two DIC images that result. It is possible for the two, wavelength multiplexed DIC images to differ in either or both shear direction or magnitude. These approaches include (1) two DIC microscopes, each operating at a different wavelength, but which share a single objective through a beam splitter; (2) a segmented DIC prism that is made in four sections where opposite sections are paired and have the same shear direction and amount, and each pair of sections have filters transmitting different wavelengths; (3) a segmented DIC prism that is located in or near an aperture stop or pupil of said DIC microscope to obtain data in two shear directions that is multiplexed by wavelength; (4) a dual field-of-view optical system with two DIC prisms, one in each path to wavelength multiplex shear direction or shear magnitude through said objective; (5) demultiplexing wavelength multiplexed DIC images through the use of a wavelength selective beam splitter and two detectors; (6) demultiplexing wavelength multiplexed DIC images through the use of a wavelength controlled source and a single detector; and (7) demultiplexing wavelength multiplexed DIC images through the use of dual field-of-view optics and a single detector. These various approaches permit rapid, robust measurement of slope in two directions. Further, phase shifting and DIC microscopy are limited to measurements within the depth of focus (DOF) of the objective while WLI microscopy is not.

What is claimed is:  
     
         1 . A differential interference contrast (DIC) microscope system comprising: 
 (a) an illumination source for illuminating a sample;    (b) a lens system for viewing the illuminated sample, including an objective, defining an optical axis;    (c) at least one detector system for receiving a sample image;    (d) mechanisms for wavelength multiplexing the shear direction or shear magnitude or both on the sample and demultiplexing the resultant DIC images on the detector; and    (e) a mechanism for modulating the phase of the DIC image.    
     
     
         2 . The DIC microscope system of  claim 1  further including a mechanism for processing the received sample image.  
     
     
         3 . The DIC microscope system of  claim 1  comprising two DIC microscopes, each operating at a different wavelength, but which share a single objective through a beam splitter.  
     
     
         4 . The DIC microscope system of  claim 3  wherein the beam splitter is based on amplitude splitting.  
     
     
         5 . The DIC microscope system of  claim 3  wherein the beam splitter is wavelength selective.  
     
     
         6 . The DIC microscope system of  claim 1  wherein the wavelength multiplexing mechanism comprises a segmented DIC prism that is made in four sections where opposite sections are paired and have the same shear direction and amount, and each pair of sections have filters transmitting different wavelengths.  
     
     
         7 . The DIC microscope system of  claim 6  wherein the sections are nominally of equal area.  
     
     
         8 . The DIC microscope system of  claim 6  wherein the wavelength filters are narrow-band thin-film filters.  
     
     
         9 . The DIC microscope system of  claim 1  wherein the wavelength multiplexing mechanism comprises a segmented DIC prism that is located in or near an aperture stop or pupil of the DIC microscope to obtain data in two shear directions that is multiplexed by wavelength.  
     
     
         10 . The DIC microscope system of  claim 1  further comprising a dual field-of-view optical system with two DIC prisms, one in each path to wavelength multiplex shear direction or shear magnitude or both through the objective.  
     
     
         11 . The DIC microscope system of  claim 1  further comprising a mechanism for demultiplexing wavelength multiplexed DIC images through the use of a wavelength selective beam splitter and two detectors.  
     
     
         12 . The DIC microscope system of  claim 1  further comprising a mechanism for demultiplexing wavelength multiplexed DIC images through the use of a wavelength controlled source and a single detector.  
     
     
         13 . The DIC microscope system of  claim 12  wherein the wavelength controlled source uses a filter wheel.  
     
     
         14 . The DIC microscope system of  claim 12  wherein the wavelength controlled source uses at least two sets of different color light emitting diodes (LEDs) that can be turned on separately.  
     
     
         15 . The DIC microscope system of  claim 12  wherein the wavelength controlled source is accomplished by selecting a different colored laser.  
     
     
         16 . The DIC microscope system of  claim 1  further comprising a mechanism for simultaneous acquisition of slope data in two directions or in two magnitudes or both through wavelength multiplexing and demultiplexing of shear direction and two detectors.  
     
     
         17 . The DIC microscope system of  claim 1  further comprising a mechanism for sequential acquisition of slope data in two directions or in two magnitudes or both through the use of a wavelength controlled source and a single detector.  
     
     
         18 . The DIC microscope system of  claim 1  further comprising a mechanism for simultaneous acquisition of slope data in two directions or in two magnitudes or both through the use of a dual field-of-view (DFOV) optical system to create two displaced images of the field-of-view on a single detector, with a projected field stop.  
     
     
         19 . A differential interference contrast (DIC) microscope system comprising: 
 (a) an illumination source for illuminating a sample;    (b) a lens system for viewing the illuminated sample, including an objective, defining an optical axis;    (c) at least one detector system for receiving a sample image;    (d) a mechanism for modulating the phase of the DIC image; and    (e) a virtual reference surface stored in a computer provided with an image capture device, the virtual reference surface being a function of the focal position of the objective.    
     
     
         20 . The DIC microscope system of  claim 19  further including 
 (f) a mechanism for processing the received sample image.  
 
     
     
         21 . The DIC microscope system of  claim 19  wherein the virtual reference: 
 (a) is represented as a table that is a function of pixel coordinate,  
 (b) two or more said tables are stored for two or more different focal positions for each shear direction and applied to slope data or a single table is stored and applied to surface data after integration of slope data to a surface, and  
 (c) the correction is interpolated for a current focal position.  
 
     
     
         22 . The DIC microscope system of  claim 19  wherein the virtual reference: 
 (a) is represented as a polynomial that is a function of pixel coordinate,  
 (b) coefficients of the polynomials are stored for two or more different focal positions for each shear direction and applied to the slope data or a single table is stored and applied to the surface data after integration of slope data to a surface, and  
 (c) the correction is interpolated for the current focal position.  
 
     
     
         23 . The DIC microscope system of  claim 19  wherein the virtual reference: 
 (a) is represented as a combination of a polynomial and a table that is a function of pixel coordinate,  
 (b) coefficients of the polynomials and tables are stored for two or more different focal positions for each shear direction and applied to the slope data or a single table is stored and applied to the surface data after integration of slope data to a surface, and  
 (c) the correction is interpolated for the current focal position.  
 
     
     
         24 . The DIC microscope system of  claim 19  wherein the virtual reference: 
 (a) is represented as a polynomial for each pixel coordinate and is a function of focal position, and  
 (b) coefficients of the polynomials are stored for each pixel for each shear direction and applied to the slope data or a single table is stored and applied to the data after integration to a surface.  
 
     
     
         25 . The DIC microscope system of  claim 19  further including a liquid crystal device (LCD) as a phase modulation device.  
     
     
         26 . The DIC microscope system of  claim 19  further including addition of a beam splitter so that the objective and a DIC prism assembly, comprised of a multiplexed DIC prism or DFOV optics with two prisms, can move as a rigid unit to permit focusing on a sample so that the relative positions of the DIC prism or prisms and objective are fixed.  
     
     
         27 . The DIC microscope system of  claim 26  further including addition of a mirror and wavelength selective beam splitter to permit the prisms and objectives to be fixed relative to each other and maximize light throughput.