Fluorescence digital image-based utilizing region of interest detection: a novel approach for chemical data interpretation in digital imaging

Abstract

Background

Digital camera-based analysis has become an important tool in the scientific field, where colour model values are employed as quantitative information for calibration purposes. However, colour models exhibit limitations in dynamic range, which restricts the linear range of digital image-based methods. To address this issue, a novel analytical signal approach based on emission areas in digital images has been developed. This new method, termed fluorescence digital image-based analysis with region of interest detection (FDIB-ROID), is reported here for the first time for analyte determination.

Results

The FDIB-ROID method achieved a linear relationship between the signal and concentration by employing emission analytes across the entire visible spectrum using quinine (QN), rhodamine 6G (RDA-6G), rhodamine B (RDA-B), and 4-Dicyanomethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM). This approach was discovered using new portable equipment built by 3D printing, with excitation in the ultraviolet to visible region (from 300 to 800 nm) and detectability similar to that of a commercial spectrofluorometer. The concentration range obtained for QN was from 50 ng mL^-1 to 1000 ng mL^-1, with a detection limit of 10 ng mL^-1 and a linear regression with an R² of 0.998. Thus, the accuracy, precision, reproducibility, and sensitivity of the method were validated by measuring QN in commercial samples. The FDIB-ROID approach has been efficiently employed in results of other studies, and the limitations of the classical approach have been overcome.

Significance

This new approach provides a broader analytical signal range for digital image-based methods. It overcomes the inherent limitations of relying solely on colour model values as analytical signals. A deeper understanding of ROID enables the design and development of novel tools that offer enhanced sensitivity and an extended dynamic working range.