Abstracts

Fluorescence and bio/chemiluminescence are widely utilized in analytical methods within biotechnology and medical fields. Generally, luminescence signals from samples are measured as relative values. However, acquiring absolute luminescence signal values, along with spectral data, provide crucial insights into the comprehensive insights into the characteristics of light emission from luminescent materials and substances. Due to the diffuse nature of light emission from samples, the "integrating sphere spectrometer" technique proves to be suitable for measuring absolute light values, expressed in units of energy (W) or number of photons. Calibration of the instrument's absolute sensitivity can be achieved using a standard lamp with traceability to national standards. Presented here are examples of absolute luminescence measurements. Firstly, quantum yield values of bioluminescence reactions were investigated using various luciferases from distinct species of firefly and glow worm. Moreover, absolute light measurement techniques contribute to establishing reference light sources for bioanalysis applications. By utilizing a stabilized LED as a reference light source, bioimaging data were collected in absolute value, resulting in more precise data comparison. The utilization of optical reference, including a reference light source, can significantly enhance reliability of bioanalysis applications, even if not absolutely calibrated but reproducible. Hence, the incorporation of optical references is recommended for bioanalysis applications within the framework of international standardization (ISO).