On the origin and correction for inner filter effects in fluorescence Part I: primary inner filter effect-the proper approach for sample absorbance correction
- 1 July 2020
- journal article
- research article
- Published by IOP Publishing in Methods and Applications in Fluorescence
- Vol. 8 (3), 033002
- https://doi.org/10.1088/2050-6120/ab947c
Abstract
Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.Funding Information
- European Social Fund (Operational Program Knowledge Education Development (POWR.03.05. 00-00-Z310/17)
This publication has 20 references indexed in Scilit:
- Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arraysNature Nanotechnology, 2017
- Introduction to FluorescencePublished by Taylor & Francis Ltd ,2014
- Synchronous fluorescence spectrometry: Conformational investigation or inner filter effect?Journal of Luminescence, 2013
- Molecular FluorescencePublished by Wiley ,2012
- Quantifying the contributions of inner-filter, re-absorption and aggregation effects in the photoluminescence of high-concentration conjugated polymer solutionsJournal of Luminescence, 2008
- Fluorescence Absorbance Inner-Filter Decomposition: The Role of Emission Shape on Estimates of Free Ca2+ Using Rhod-2Applied Spectroscopy, 2007
- Probing Protein Folding and Conformational Transitions with FluorescenceChemical Reviews, 2006
- Principles of Fluorescence SpectroscopyPublished by Springer Science and Business Media LLC ,2006
- Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ BindingBiophysical Journal, 2003
- Correction for light absorption in fluorescence studies of protein-ligand interactionsAnalytical Biochemistry, 1983