Abstract

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.

Digital holographic microscopy (DHM) is an interferometric technique with several advantages over traditional light microscopy. A hologram is an interference pattern produced by a coherent "reference" beam recombining with an "object" beam from the same source but that has passed through a sample (Fig. 1). The interference fringes encode the phase and amplitude of the object beam at the detector and can be used to calculate their values at any point along the path of the object beam (e.g. within the sample volume). These values can be used to calculate an intensity image (equivalent to brightfield) and a "phase" image that displays the index of refraction times the thickness of the objects of that index along the path. Resolution is the same as a conventional optical microscope with a comparable objective, but for typical optics needed to image prokaryotes, no loss in resolution is seen with samples as thick as a millimeter. This represents an approximately 100-fold improvement in depth of field over high-power light microscopy. No focusing is required and there are no moving parts. Plane-by-plane images are obtained through numerical reconstruction [1, 2]; all z-plane information is encoded in a single hologram.