Abstract
Digital holographic microscopy (DHM) is an emerging imaging technique that permits instantaneous capture of a relatively large sample volume. However, large volumes usually come at the expense of lower spatial resolution, and the technique has rarely been used with prokaryotic cells due to their small size and low contrast. In this paper we demonstrate the use of a Mach-Zehnder dual-beam instrument for imaging of labeled and unlabeled bacteria and microalgae. Spatial resolution of 0.3 μm is achieved, providing a sampling of several pixels across a typical prokaryotic cell. Both cellular motility and morphology are readily recorded. The use of dyes provides both amplitude and phase contrast improvement and is of use to identify cells in dense samples.
Highlights
AND BACKGROUNDDigital holographic microscopy (DHM) is an emerging technique of relevance to fields of biology, chemistry, and physics where instantaneous sampling of a three-dimensional volume is desired
All of these dyes’ fluorescence is emitted in the green (Cerulean), orange (NanoOrange), or red (ZnTPP) and does not alter DHM imaging, as the fluorescence signal is both temporally incoherent and comparatively very weak relative to the excitation band, though it can serve as a useful confirmation of successful labeling using fluorescence microscopy in parallel
With V. alginolyticus, cell bodies were strongly labeled with NanoOrange, and sufficient dye was bound to flagella to enable their visualization under ordinary widefield epifluorescence microscopy (Figure 2B)
Summary
AND BACKGROUNDDigital holographic microscopy (DHM) is an emerging technique of relevance to fields of biology, chemistry, and physics where instantaneous sampling of a three-dimensional volume is desired. The optical components involved in DHM are relatively simple and low-cost compared to those needed for fluorescence microscopy. Because most of the illuminating photons are captured, low-power excitation sources can be used— milliwatt diode lasers, which are available in a large range of wavelengths. Because a single wavelength of illumination light is used, compound objective lenses with chromatic aberration correction are not needed. Low cost instruments may be constructed using web cameras, light-emitting diode illumination, and simple aspheric lenses (Lu et al, 2014; Mico et al, 2014). While DHM is usually used in transmission mode for imaging through low-density samples, it is possible to build instruments with similar components to operate in reflectance mode (Colomb et al, 2010; Lee et al, 2011; Yaqoob et al, 2011)
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