Högskolan i Skövde

Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide (NADH) offers a label-free approach to map cellular metabolism. Unfortunately, its value is hindered by the lack of characterisation of uncertainties in measurement, polarisation artefacts, and environmental variables such as temperature, viscosity, and pH to which fluorescence lifetime is sensitive. To account for this, measurements of fluorescence intensity decays of NADH under controlled environmental conditions were performed. Systematic changes in pH, temperature, and viscosity were followed, and their effects characterised. The importance of the correct management of polarisation artefacts that could affect lifetime estimation was also addressed with the study of the angle of an emission polariser. It was possible to assess statistical significance between central tendency parameters due to a thorough examination of sources of error. Significant differences were observed for all of the tracked environmental conditions except for pH (p<0.05), indicating that temperature and viscosity play a role in shifting fluorescence lifetime for NADH, whereas pH does not. Lastly, the importance of correct error propagation was underlined, since, despite the instrument response function (IRF) contributing the most to uncertainty (~0.06 ns), error propagation methods that accounted for parameter correlation (Monte Carlo) yielded statistical significance where a broader approach (Taylor first-order approximation) would not. All these findings help better understand NADH fluorescence and can be applied to FLIM measurements.