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Published on: September 22, 2019
MTT assay analyzer: an open-source smartphone-based web application for semi-quantitative colorimetric analysis of
Jana Pokorná1, Vladan Bernard1
1Department of Biophysics, Faculty of Medicine, Masaryk University, Brno, Czechia.
Objective:
The MTT assay is usually used to evaluate cell viability and metabolic activity, typically quantified via spectrophotometric absorbance. Conventional plate readers are reliable but costly and inaccessible in many educational and resource-limited laboratories. Consumer smartphones offer a low-cost alternative, but existing methods require dedicated hardware or manual processing.
Methods:
MTT assay analyzer is an open-source, mobile-optimized web application for semi-quantitative analysis of MTT assay photographs taken with smartphones, tested on iPhone Air, processed directly in a standard browser without installation. The workflow includes manual alignment to two reference points, RGB colour sampling with spatial averaging, grayscale pseudo-absorbance estimation, and gamma-green channel corrected absorbance via power-law linearization. Optional 2-4-point linear calibration uses spectrophotometer references. Validation was performed in five independent experiments using Pearson correlation, Bland-Altman analysis, and root mean square error (RMSE) against a spectrophotometer.
Results:
A total of 372 paired measurements were analysed across five independent experiments (absorbance range 0.01-1.78 AU). Across all experiments, the grayscale method with two-point calibration (GS + Cal) achieved the highest agreement with spectrophotometric reference values (pooled: r = 0.991, R² = 0.983, RMSE = 0.057 AU, 95% LoA [-0.124, +0.090]). The uncalibrated grayscale method showed systematic underestimation (pooled mean bias -0.052 AU), which was substantially reduced by calibration (mean bias -0.017 AU). Per-experiment RMSE for GS + Cal ranged from 0.035 to 0.078 AU. Pearson r ranged from 0.924 to 0.999 across all methods and experiments, with the lowest values observed in experiments with a restricted dynamic range (maximum absorbance ≤ 0.95 AU) rather than reflecting measurement failure. Two-point calibration reduced RMSE in all five experiments.
Conclusions:
MTT assay analyzer delivers a low-cost tool for smartphone-based semi-quantitative MTT analysis, ideal for trend detection, screening, and education where spectrophotometers are unavailable.
