Digital image processing based fluorescent signal validation and calibration

The fluorescent intensities of microscopic images were analysed using image processing software, ImageJ2 bundled with 64-bit Java 1.8.0_112 [35]. Briefly, three isolated single cells were selected from each image, and cell boundaries were marked with the freedom selection tool and then retrieved the Area, Mean Gray Value, Minimum/Maximum Gray Values, and Integrated Density. The Gray values were measured simultaneously for the immediate background of each cell. The calculated mean intensity values were then converted into Corrected Total Cell Fluorescent (CTCF) values by applying the equation described elsewhere [36]. The average CTCF values of all biological replicates were tabulated corresponding to the calibration graphs.

The capability of E.coli-BL21(DE3)-pJET1.2-CadA/CadR cells to fluoresce significant signals under optimum pH conditions were examined by plotting the graph between pH values (2 – 12 pH) vs CTCF values. A single colony of E. coli-BL21 harbouring pJET1.2-CadA/CadR plasmid was grown overnight at 37 °C and pH in the medium 7.0. The overnight culture was diluted tenfold in minimal (M9) medium supplemented with 100 μg/mL ampicillin and incubated with 2.0 ppb of Cd²⁺, Zn²⁺, Pb²⁺, and Ni²⁺ in the medium in an orbital shaker at 200 rpm with a pH gradient from 2 – 12 pH for 2 h optimal incubation time and at the constant pH value of 7.0. The cells were subjected to fluorescent microscopy as described in section "Fluorescent microscopy" and image processing was done as described in section "Digital image processing" to quantify the pH dependent biosensing signals. Triplicate measurements were obtained for each sample and the mean value was taken.

The capacity of E.coli-BL21(DE3)-pJET1.2-CadA/CadR cells to fluoresce significant signals in optimum temperature conditions was examined by plotting the graph between temperature of the medium (0 – 60 °C) vs CTCF values. A single colony of E. coli-BL21 harbouring pJET1.2-CadA/CadR plasmid was grown overnight at 37 °C in pH of the medium 7.0. The overnight culture was diluted tenfold in minimal (M9) medium supplemented with 100 μg/mL ampicillin and incubated with 2.0 ppb concentrations of Cd²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ in the medium in an orbital shaker at 200 rpm with a temperature gradient of 0, 25, 37, 45 and 60 °C with 2 h of optimal incubation time. The cells were subjected to fluorescent microscopy as described in section "Fluorescent microscopy" and image processing was done as described in section "Digital image processing" to quantify the temperature dependent biosensing signals. Triplicate measurements were obtained for each sample and the mean values were taken.

The effect of heavy metal incubation time on the intensity of fluorescent signals were monitored by conducting Cd²⁺, Zn²⁺ and Pb²⁺ treatments at different time points of 0, 1, 2, 3, 4, 5, 6 and 7 h with the concentration gradient of 1, 2, 3, 4, 5 and 6 ppb. The fluorescent intensity data converted into CTCF values from each time point were plotted on 3 dimensional plots with the Z-axis representing fluorescent intensities in CTCF, the Y-axis representing heavy metal concentration in ppb and the X- axis representing Time (hours). In generating the plots, the multiple experimental data was saved in the table format in three separate.csv files dedicated for Cd²⁺, Pb²⁺ and Zn²⁺. To visualize the data in 3-Dimentions (3D), Jupyter Notebook IDE v6.1.1 (https://jupyter.org/) was used. The code was implemented in Python v3.7.7 (https://www.python.org/downloads/release/python-377/). Initially,.csv files (Comma Separated Values) containing data were loaded into data frames (https://pandas.pydata.org/pandas-docs/stable/reference/frame.html) for visualization using Pandas library v1.1.0 (https://pandas.pydata.org/). Subsequently, using Plotly library v4.9.0 (https://plotly.com/), data in data frames was visualized in 3D Surface Plots.