1. Zetsche, A. El Mallahi, F. Dubois, C. Yourassowsky, J. C. Kromkamp, F.J.R. Meysman,

Limnol. Oceanogr.: Methods 12, 2014, 757-775 © 2014, by the American Society of Limnology and Oceanography, Inc.

Nanoplanktonic cells similar in shape were successfully detected and classified from images captured with an off-axis digital holographic microscope with partial coherence and a flow-through system based at the Université Libre de Bruxelles (Belgium). Morphological and textural features of light intensity images were extracted, as well as textural features of the phase information images, unique to DHM. An overall classification score of 92.4% demonstrated the potential of holographic-based imaging-in-flow to replace flow cytometry and classical brightfield microscopy for the detection of similar looking organisms in the nanoplankton range.

A qMod mounted on a Zeiss Axioplan was used in this study to observe changes of internal cell structures in one of the nanoplanktonic organisms, Chlorella autotrophica, as growth conditions for the culture changed. Phosphate depletion over time in the culture significantly affected the physiology of the cells. Cell detection and feature extraction of images captured with the qMod confirmed that changes occurred within the cells over time, yet that these were minor compared to the differences observed between the three different species. This reiterated the ability of DHM to detect cellular changes and to differentiate species based on the added information gained from phase images.

Figure: False-color rendition of the phase information (optical thickness) from holograms captured with the qMod of (A) cells of the green algae  Chlorella autotrophica  imaged on day 2 of the phosphate free culturing conditions compared to (B) a cell imaged on day 9 of the experiment when cell physiology was significantly impaired. (Images courtesy of E. Zetsche, unpubl.).

Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time-consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on “imaging-in-flow,” i.e., the ability to acquire microscopic images of planktonic cells in a flow-through mode. Imaging-in-flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e.g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging-in-flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off-axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape-based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic-based imaging-in-flow for similar looking organisms in the nanoplankton range. © 2014, by the American Society of Limnology and Oceanography, Inc.

El Mallahi, A., Minetti, C., & Dubois, F. (2013). Automated three-dimensional detection and classification of living organisms using digital holographic microscopy with partial spatial coherent source: Application to the monitoring of drinking water resources. Applied Optics, 52(1), A68-A80.

Yourassowsky, C., & Dubois, F. (2014). High throughput holographic imaging-in-flow for the analysis of a wide plankton size range. Optics Express, 22(6), 13. doi:DOI:10.1364/OE.22.006661