@article{
 title = {mhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy},
 type = {article},
 year = {2009},
 identifiers = {[object Object]},
 keywords = {Algorithms,Calibration,Cell Line,Coloring Agents,Coloring Agents: chemistry,Energy-Transfer,Flim,Fluorescence,Fourier Analysis,Frequency-Domain,Humans,Imaging Microscopy,Living Cells,Microscopy,Microscopy: instrumentation,Microscopy: methods,Phase,Solutions,System,Time Factors,Tumor},
 pages = {1557-1570},
 volume = {17},
 websites = {http://www.ncbi.nlm.nih.gov/pubmed/19188985,http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=000263432400044,file://localhost/Users/cfk23/Documents/Papers/2009/Schlachter/Opt Express 2009 Schlachter.pdf,papers://5b342310-4c1b-4925-bbf8-38f14091331d/Paper/p51},
 month = {1},
 day = {2},
 city = {Univ Cambridge, Dept Chem Engn & Biotechnol, Cambridge CB2 1RA, England},
 id = {7e30e62c-faaf-31b1-beb4-0479345bce7d},
 created = {2010-07-07T09:21:00.000Z},
 file_attached = {true},
 profile_id = {a836303d-69f8-302f-a6cc-6bab5a6094d9},
 last_modified = {2013-11-12T12:24:30.000Z},
 read = {false},
 starred = {false},
 authored = {true},
 confirmed = {true},
 hidden = {false},
 source_type = {JOUR},
 abstract = {Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition. (C) 2009 Optical Society of America},
 bibtype = {article},
 author = {Schlachter, S and Elder, undefined and Esposito, undefined and Kaminski, undefined and Frank, undefined and Geest, Van and Elder, a D and Esposito, A and Kaminski, G S and Frank, J H and van Geest, L K and Kaminski, C F},
 journal = {Optics Express},
 number = {3}
}

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However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition. 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