Optical determination of intracellular water in apoptotic cells. (30th October 2013)

• Record Type: Journal Article
• Authors: Model, Michael A.; Schonbrun, Ethan
• Is Part Of: Journal of physiology. Volume 591:Number 23(2013:Dec.)
• Page Start: 5843
• Page End: 5849
• Publication Date: 2013-10-30
• Subjects: Physiology -- Periodicals; 612.005
• DOI: 10.1113/jphysiol.2013.263228
• Languages: English
• ISSNs: 0022-3751

Abstract

Key points: Determination of intracellular water can be achieved by a combination of brightfield and transmission‐through‐dye imaging, all realized on a standard transmission microscope. The method permits sensitive detection of water loss in cells exposed to apoptotic agents. Cell water measurements are not affected by separation of apoptotic bodies and can be applied to samples where measurements of cell volume alone would be insufficient. Abstract Intracellular water plays a critical role in apoptotic and necrotic cell death. We describe a method for quantifying cell water by application of two previously described variants of transmission microscopy. By taking two axially displaced brightfield images, the phase shift of the transmitted wave was computed using the transport‐of‐intensity equation. At the same time, cell thickness was determined by transmission through an externally applied dye ('transmission‐through‐dye' microscopy); switching between these two imaging modalities was accomplished by simply changing the illumination wavelength. The sets of data thus obtained allow computation of the refractive index and cell water content within individual cells. The method was illustrated using cells treated with apoptotic agents staurosporine and actinomycin D and with necrosis inducer ionomycin. Water imaging allows discrimination between apoptotic volume decrease due to dehydration from that due to detachment of apoptotic bodies and can be used on samples where cell volume determination alone would be difficult or insufficient.