Two postprocessing techniques for the elimination of background autofluorescence for fluorescence lifetime imaging microscopy.
The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
Jones
Phillip B
PB
Harvard Medical School, Massachusetts General Hospital, MassGeneral Institute for Neurodegenerative Disorders, 114 16th Street, Charlestown, Massachusetts 02129, USA.
Rozkalne
Aneta
A
Meyer-Luehmann
Melanie
M
Spires-Jones
Tara L
TL
Makarova
Alexandra
A
Kumar
Anand T N
AT
Berezovska
Oksana
O
Bacskai
Brian B
BB
Hyman
Bradley T
BT
D000465:Algorithms; D000818:Animals; D016477:Artifacts; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013382:Subtraction Technique