On the origin and correction for inner filter effects in fluorescence. Part II: secondary inner filter effect -the proper use of front-face configuration for highly absorbing and scattering samples.

Fluorescence is an established technology for studying molecular processes and molecular interactions. More recently fluorescence became a leading method for detection, sensing, medical diagnostics, biotechnology, imaging, DNA analysis, and gene expression. Consequently, precise and accurate measurements in various conditions have become more critical for proper result interpretations. Previously, in Part 1, we discussed inner filter effect type I, which is a consequence of the instrumental geometrical sensitivity factor and absorption of the excitation. In this part, we analyze inner filter effect type II and discuss the practical consequences for fluorescence measurements in samples of high optical density (absorbance/scattering). We consider both the standard square and front-face experimental configurations, discuss experimental approaches to limit/mitigate the effect and discuss methods for correcting and interpreting experimental results.