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Described over 100 years ago, the Gouy phase anomaly refers to the additional phase shift that is accumulated as a wave passes through focus. It is potentially useful in analyzing any type of phase-sensitive imaging; in light microscopy, digital holographic microscopy (DHM) provides phase information in the encoded hologram. One limitation of DHM is the weak contrast generated by many biological cells, especially unpigmented bacteria. We demonstrate here that the Gouy phase anomaly may be detected directly in the phase image using the z-derivative of the phase, allowing for precise localization of unlabeled, micrometer-sized bacteria. The use of dyes that increase phase contrast does not improve detectability. This approach is less computationally intensive than other procedures such as deconvolution and is relatively insensitive to reconstruction parameters. The software is implemented in an open-source FIJI plug-in.more » « less
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Following the previous article, here we describe the first field demonstration of the ELVIS system, performed at Newport Beach, CA. We examined ocean water to detect microorganisms using the combined holographic and light-field fluorescence microscope and successfully detected both eukaryotes and prokaryotes. The shared field of view provided simultaneous bright-field (amplitude), phase, and fluorescence information from both chlorophyll autofluorescence and acridine orange staining. The entire process was performed in a nearly autonomous manner using a specifically designed sample processing unit (SPU) and custom acquisition software. We also discuss improvements to the system made after the field test that will make it more broadly useful to other types of fluorophores and samples.more » « less
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This is the first of two articles on the Extant Life Volumetric Imaging System (ELVIS) describing a combined digital holographic microscope (DHM) and a fluorescence light-field microscope (FLFM). The instrument is modular and robust enough for field use. Each mode uses its own illumination source and camera, but both microscopes share a common objective lens and sample viewing chamber. This allows correlative volumetric imaging in amplitude, quantitative phase, and fluorescence modes. A detailed schematic and parts list is presented, as well as links to open-source software packages for data acquisition and analysis that permits interested researchers to duplicate the design. Instrument performance is quantified using test targets and beads. In the second article on ELVIS, to be published in the next issue of Microscopy Today , analysis of data from field tests and images of microorganisms will be presented.more » « less
