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001-es BibID:	BIBFORM109070
035-os BibID:	(cikkazonosító)2934 (Scopus)85148394136 (WoS)000955795600021
Első szerző:	Rebenku István
Cím:	Pixel-by-pixel autofluorescence corrected FRET in fluorescence microscopy improves accuracy for samples with spatially varied autofluorescence to signal ratio / Rebenku István, Lloyd Cameron B., Szöllősi János, Vereb György
Dátum:	2023
ISSN:	2045-2322
Megjegyzések:	The actual interaction between signaling species in cellular processes is often more important than their expression levels. Förster resonance energy transfer (FRET) is a popular tool for studying molecular interactions, since it is highly sensitive to proximity in the range of 2-10 nm. Spectral spillover-corrected quantitative (3-cube) FRET is a cost efective and versatile approach, which can be applied in fow cytometry and various modalities of fuorescence microscopy, but may be hampered by varying levels of autofuorescence. Here, we have implemented pixel-by-pixel autofuorescence correction in microscopy FRET measurements, exploiting cell-free calibration standards void of autofuorescence that allow the correct determination of all spectral spillover factors. We also present an ImageJ/Fiji plugin for interactive analysis of single images as well as automatic creation of quantitative FRET efciency maps from large image sets. For validation, we used bead and cell based FRET models covering a range of signal to autofuorescence ratios and FRET efciencies and compared the approach with conventional average autofuorescence/background correction. Pixel-by-pixel autofuorescence correction proved to be superior in the accuracy of results, particularly for samples with spatially varying autofuorescence and low fuorescence to autofuorescence ratios, the latter often being the case for physiological expression levels.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk
Megjelenés:	Scientific Reports. - 13 : 1 (2023), p. 1-15. -
További szerzők:	Lloyd, Cameron B. Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (biofizikus, orvos)
Pályázati támogatás:	GINOP-2.2.1-15-2017-00072 GINOP K135938 OTKA
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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