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Holographic And Fluorescence Microscopy. In One Single System.

Stain-Free Viability Determination

Diatom cells between standard glass microscope slides and cover slips

"Typical procedures for the determination of cell viability currently rely on staining with various fluorescent markers (Veldhuis et al. 2001)."

Dr. Zetsche and co-authors demonstrate "for the first time that differentiation between live and dead diatom cells can be made based solely on 3D image analysis procedures without any preceding staining procedures." It confirms "the potential of DHM instrumentation to improve our understanding of morphological, ecological, as well as physiological aspects of diatoms."

Ovizio's technology achieves this by recording both the amplitude as well as the quantitative phase information of a sample (see Hobson & Watson 2002, Di Caprio et al. 2012, Dubois et al. 2006). The phase information reveals differences in the optical path length (OPL) through an object, and is therefore in essence a measurement of how photons are slowed down differently in various parts of the object. As a result, it can detect subtle changes in the OPL within a single cell, or variations in OPL between different cells.

Figure: DHM phase information and viability. Representative examples of individual cells from two diatom cultures were imaged in their natural healthy state: (a) Navicula sp. and (d) Nitzschia cf. pellucida. Subsequent heat-killing shows changes in the OPL landscape of the cells for (b) Navicula sp. and (e) N. cf. pellucida. The white scale bar applies to all four images. (c) and (f) show OPL profiles drawn across the longest axes of the cells as exemplified with the white stippled lines. (Taken from Zetsche et al. 2016)

See also:
VELDHUIS M.J.W., KRAAY G.W. & TIMMERMANS K.R 2001. Cell death in phytoplankton: correlation between changes in membrane permeability, photosynthetic activity, pigmentation and growth. European Journal of Phycology 36: 167-177.

HOBSON P.R. & WATSON J. 2002. The principles and practice of holographic recording of plankton. Journal of Optics A: Pure and Applied Optics 4: S34-S49.

DI CAPRIO G., COPPOLA G., DE STEFANO L., DE STEFAN M., ANTONUCCI A., CONGESTRI R.&DE TOMMASI E. 2012. Shedding light on diatom photonics by means of digital holography. Journal of Biophotonics 7: 341-350.

DUBOIS F., YOURASSOWSKY C., MONNOM O., LEGROS J.-C., DEBEIR O., VAN HAM P., KISS R.&DECAESTECKER C. 2006. Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration. Journal of Biomedical Optics 11(5): 054032.

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