Eva-Maria Zetsche, Ahmed El Mallahi & Filip J. R. Meysman. Diatom Research, 31:1, 1-16, 2016.

A recent publication by Zetsche et al. (2016) highlights the difficulties in imaging aquatic organisms such as diatoms: “Diatom cells are for the large part transparent, and since transparent substances or objects, by definition, do not absorb light in appreciable quantities when suspended in water, these entities are hard to discriminate and detect by microscopy techniques that rely on intensity information alone”.

“Piper (2011) suggested that interference-based contrast microscopy reveals the shape and structure of cells more clearly, as it improves the plasticity and contour sharpness.” Digital holographic microscopy (DHM) is in fact an interference-based approach and Dr. Zetsche and her co-authors are able to show that with DHM “the structural organization of diatoms is more clearly determined, in terms of cellular components, shapes and features.”

Ovizio’s qMod, a differential digital holographic microscopy camera for classical microscopes, is one of the instruments which offers a tool for the improved discrimination of living and dead diatoms (success rate >95%). Possible future applications can be the live-dead discrimination of microscopic aquatic organisms, as well as improved species identification. “Certain species of diatoms are frequently used to assess the water quality of rivers and lakes as well as coastal areas (Anton-Garrido et al. 2013, Kelly et al. 2009, Sabater et al. 2007). DHM may facilitate the live-dead differentiation of cells and thus improve these monitoring procedures” (Zetsche et al. 2016).

Figure: (a) Hologram of a cleaned frustule of  Stauroneis  sp. (University of Gent, Belgium) as obtained with an Ovizio digital holographic microscope. This hologram contains both light-intensity information (b) as well as phase information (c) representing the optical path length (OPL) of the object. (d) The OPL of an object is more clearly visualized with false coloring of the phase information. (Taken from Zetsche et al. 2016)

Recent advances in optical components, computational hardware and image analysis algorithms have led to the development of a powerful new imaging tool, digital holographic microscopy (DHM). So far, DHM has been predominantly applied in the life sciences and medical research, and here, we evaluate the potential of DHM within a marine context, i.e. for studying the morphology, physiology and ecology of diatoms. Like classical light microscopy, DHM captures light-intensity information from objects, but in addition, it also records the so-called phase information. Because this phase information is recorded in a fully quantitative way, it gives access to a whole new type of image properties, which suitably extend the range of microscopy applications in diatom research. Here, we demonstrate the ability of DHM to provide structural information on internal cell organelles as well as the silica frustules of diatoms. By combining the light intensity and phase information, one also obtains the optical ‘fingerprint’ of a cell, which can be used to discriminate between cells of separate diatom species or to differentiate between living and dead cells (as demonstrated here for two diatom species Navicula sp. and Nitzschia cf. pellucida). Finally, we use chains of Melosira sp. to demonstrate the capacity of DHM to refocus post-acquisition, and combine holograms with fluorescent images, and the ability of DHM to image transparent substances, such as extracellular polymeric substances. Overall, DHM is a promising versatile microscopic technique, allowing diatoms to be investigated in vivo, over time, without the need for staining, and quantitatively in terms of their phase information. Thus, DHM can provide new insights into the structure, as well as the physiology and ecology of diatoms.

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