TY - JOUR
T1 - On the origin and correction for inner filter effects in fluorescence Part I
T2 - Primary inner filter effect-the proper approach for sample absorbance correction
AU - Kimball, Joseph
AU - Chavez, Jose
AU - Ceresa, Luca
AU - Kitchner, Emma
AU - Nurekeyev, Zhangatay
AU - Doan, Hung
AU - Szabelski, Mariusz
AU - Borejdo, Julian
AU - Gryczynski, Ignacy
AU - Gryczynski, Zygmunt
AU - Gryczynski, Zygmunt
N1 - Publisher Copyright:
© 2020 IOP Publishing Ltd.
PY - 2020/7
Y1 - 2020/7
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85085904462&partnerID=8YFLogxK
U2 - 10.1088/2050-6120/ab947c
DO - 10.1088/2050-6120/ab947c
M3 - Article
C2 - 32428893
AN - SCOPUS:85085904462
SN - 2050-6120
VL - 8
JO - Methods and applications in fluorescence
JF - Methods and applications in fluorescence
IS - 3
M1 - 033002
ER -