James Rayburn, Biology
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ImageJ is a free computer program that processes java-based images. This program was created at the National Institute of Health and the laboratory for optical and computational instrumentation. ImageJ contains multiple plugins for processing and analyses. The particular plugin for this test is called ReadPlate. ReadPlate measures the absorbance of an image of a multi-well plate up to 96 wells. This is useful if you do not have immediate access to a commercially built plate reader. This experiment was intended to determine the differences of absorbance efficiency of a spectrophotometer and picture analysis from the ImageJ ReadPlate plugin. To test the accuracy of the ImageJ Plugin, comparisons with a spectrophotometer were made using concentrations of methylene blue. In addition, comparisons to a Bradford protein analysis was performed. Concentrations of methylene blue were analytically prepared that ranged from (0.001-0.2 mg/L). Protein standard concentrations with unknowns were prepared. Once concentrations were made, samples were placed into 3 mL cuvettes and measured on a spectrophotometer and recorded. Concentration samples were also placed into duplicate wells of a 96 well plate. The plate was placed in a light box and a picture of the plate was taken and analyzed on ImageJ. The ImageJ ReadPlate had 4 channels to measure the picture related to wavelength (color). Absorbances were then compared from the spectrophotometer and the ReadPlate plugin and the percent difference was calculated. The results indicated that the data from ImageJ using the red channel was more consistent with the absorbances from the spectrophotometer than the other channels on ImageJ with a reduction of sensitivity by ~30%. This would mean that the ImageJ ReadPlate is a viable option when a commercial plate reader is not available.
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Winter, Ansley, "Comparing the Accuracy of the ImageJ ReadPlate Plugin to the Spectrophotometer" (2020). JSU Student Symposium 2020. 23.