Automated Fluorescence Lifetime Imaging High-Content Analysis of Forster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells.

ABSTRACT: 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

SUBMITTER: Guo W 

PROVIDER: S-EPMC6537140 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature