Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data.
Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error-weighting results in a non-linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime-based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.
Clayton
A H A
AH
Ludwig Institute for Cancer Research, PO Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia.
Hanley
Q S
QS
Verveer
P J
PJ
D002232:Carbocyanines / Q000378:metabolism*; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D066246:ErbB Receptors