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Quantitative Determination of Cell Viability Using Absorbance Microscopy



Greta Babakhanova, Stephen Zimmerman, Laura Pierce, Sumona Sarkar, Nicholas Schaub, Carl Simon Jr.


Cell viability is an essential measurement for cell therapy products. One of the most common tests is Trypan blue (TB) dye exclusion where blue-stained cells are counted via imaging. Typically, live and dead cells are counted based on pixel intensities of brightfield images. Brightfield pixel intensity values vary arbitrarily making it difficult to compare results collected at different times or on different microscopes. Herein, an absorbance imaging method to determine intracellular TB molarity is demonstrated. The intensity pixels of the brightfield images are converted to absorbance maps which are subsequently used to calculate moles of TB by applying the Beer-Lambert Law. The validation of absorbance microscopy was demonstrated using neutral density filters with known optical density (OD) values. Results from four different microscopes demonstrate a mean deviation of 7.5% from the reported OD values. When assessing TB-stained Jurkat cells, TB was not detected in live cells, while heat-shock killed cells had 20.4 fmol and 21.1 fmol of TB per cell when measured with two different microscopes (3.7% difference). Cells that were killed by formaldehyde fixation had 9.2 fmol of TB per cell. This 55% drop from heat-shock to fixation cells suggests that the killing mechanism affects the amount of TB taken up by cells. Finally, absorbance imaging of 1:1 mixtures of live:dead cells resulted in dead calls 52% (S.D. 6%) of the time. Instead of making live-dead calls using arbitrary intensity values, absorbance imaging yields traceable molarity values. Traceable units can be compared, which is useful for assuring the quality of biomanufacturing processes.
PLoS One


cell viability, cell counting, trypan blue assay, absorbance microscopy, tissue engineering, cell therapy


Babakhanova, G. , Zimmerman, S. , Pierce, L. , Sarkar, S. , Schaub, N. and Simon Jr., C. (2022), Quantitative Determination of Cell Viability Using Absorbance Microscopy, PLoS One, [online],, (Accessed April 17, 2024)
Created January 19, 2022, Updated February 8, 2023