Multiwavelength digital holographic microscopy (DHM) has been used to improve phase reconstructions of digital holograms by reducing 2π phase ambiguities. However, most samples used as test images have been solid or adhered to a surface, making it easy to determine focal planes and correct for chromatic aberration. In this study we apply 3-wavelength off-axis DHM to swimming protozoa containing distinct spectral features such as chlorophyll and carotenoids. We reconstruct the holograms into amplitude and phase images using the angular spectrum method. Methods for noise subtraction, chromatic aberration correction, and image registration are presented for both amplitude and phase. Approaches to phase unwrapping are evaluated and compared to expected results from simulated holograms. The algorithms used are implemented in plug-ins using the open source Fiji platform and are available for use, significantly expanding the open-source software available for DHM.

We are developing a unique multi-modal volumetric microscope capable of instantaneous imaging of sample volumes approximately 100-fold larger than those sampled by conventional fluorescence microscopy, with spatial resolution sufficient to detect the smallest prokaryotes (Figure 1). This instrument combines two modalities for the first time: Digital Holography Microscopy (DHM) and Fluorescence Light Field Microscopy (FLFM). The instrument will be packaged for field use in aquatic environments. It will be used in the lab, and also made available to researchers inside and outside the home institutions, enabling a wide array of environmental microbiology applications.

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