Imperial College London

ProfessorPaulFrench

Faculty of Natural SciencesDepartment of Physics

Professor of Physics and Vice Dean (Research) - FoNS
 
 
 
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Contact

 

+44 (0)20 7594 7706paul.french Website

 
 
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Assistant

 

Ms Judith Baylis +44 (0)20 7594 7713

 
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Location

 

609Blackett LaboratorySouth Kensington Campus

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Summary

 

Publications

Citation

BibTex format

@article{Guo:2019:10.1177/2472630318819240,
author = {Guo, W and Kumar, S and Gorlitz, F and garcia, EC and Alexandrov, Y and Munro, I and Kelly, D and Warren, S and Thorpe, P and Dunsby, C and French, P},
doi = {10.1177/2472630318819240},
journal = {Slas Technology},
pages = {308--320},
title = {Automated fluorescence lifetime imaging high content analysis of Förster resonance energy transfer between endogenously-labeled kinetochore proteins in live budding yeast cells},
url = {http://dx.doi.org/10.1177/2472630318819240},
volume = {24},
year = {2019}
}

RIS format (EndNote, RefMan)

TY  - JOUR
AB - We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.
AU - Guo,W
AU - Kumar,S
AU - Gorlitz,F
AU - garcia,EC
AU - Alexandrov,Y
AU - Munro,I
AU - Kelly,D
AU - Warren,S
AU - Thorpe,P
AU - Dunsby,C
AU - French,P
DO - 10.1177/2472630318819240
EP - 320
PY - 2019///
SN - 2472-6303
SP - 308
TI - Automated fluorescence lifetime imaging high content analysis of Förster resonance energy transfer between endogenously-labeled kinetochore proteins in live budding yeast cells
T2 - Slas Technology
UR - http://dx.doi.org/10.1177/2472630318819240
UR - http://hdl.handle.net/10044/1/66504
VL - 24
ER -