Combining optical quadrature and differential interference contrast to facilitate embryonic cell counting with fluorescence imaging for confirmation

Abstract

The Multifunctional Staring Mode Microscope was developed to permit three modes of imaging for cell counting in mouse embryos: Optical Quadrature, Differential Interference Contrast (DIC), and Fluorescence Imaging. The Optical Quadrature Microscope, consisting of a modified Mach-Zender Interferometer, uses a 632.8 nm laser to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras, preceded by multiple beamsplitters, are used to read the four interferograms, which are then combined to produce an image of the complex electric field amplitude. The phase of the complex amplitude is then unwrapped using a 2-D phase unwrap algorithm and images of optical path length are produced. To combine the additional modes of DIC and Fluorescence Imaging with the Optical Quadrature Microscope, a 632.8 nm narrow bandpass beamsplitter was placed at the output of the microscope. This allows the laser light to continue through the Mach-Zender while all other wavelengths are reflected at 90 degrees to another camera. This was effective in combining the three modes as the fluorescence wavelength for the Hoechst stain is well below the bandpass window of the beamsplitter. Both live and fixed samples have been successfully imaged in all three modes. Accuracy in cell counting was achieved by using the DIC image for detecting cell boundaries and the Optical Quadrature image for phase mapping to determine where cells overlap. The final results were verified by Hoechst fluorescence imaging to count the individual nuclei. Algorithms are currently being refined so larger cell counts can be done more efficiently.