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. |
