Combining off-axis digital holography with fluorescence for simultaneous fluorescence and quantitative phase imaging

Off-axis digital holographic microscopy (DHM) is a microscopy technique capable of extracting and translating quantitative phase information (QPI) from the sample into 4D topographical data, which enables the study of phenotypic changes with sub-second temporal resolution. DHM has been widely implemented for long-term live-cell imaging and microelectromechanical systems characterization. However, as a bright field technique, relying on DHM imaging alone can be challenging especially in characterizing sub-cellular structures. Here we report on the design and implementation of a DHM module that can be retrofitted into a commercial dual-deck Olympus IX83 microscope base, transforming the inverted microscope into a hybrid DHM and fluorescence microscopy (DHM-FM) system. In order to achieve simultaneous acquisition of DHM and fluorescence data, the DHM module is designed to integrate illumination, dichroic filter, and interference submodules. The condenser of the microscope was replaced by the DHM illumination module, and the dichroic module was placed in the microscope platform to separate the DHM beam from the fluorescence signal. Finally, the DHM signal was delivered to the interference module outside the microscope body. As the DHM and fluorescence subsystems share the same field of view, this platform can acquire complementary data simultaneously. The capabilities of this coupled platform were explored through a series of pilot studies, including examining phase condensate formation and cellular dynamics. This modular configuration will help others explore the potential of off-axis DHM in their own work and, in particular, the potential of coupled DHM-fluorescence imaging.