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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
Published on: January 18, 2017
Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse
B Treanor1, P M P Lanigan, K Suhling
1Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. b.treanor@imperial.ac.uk
Insights
Fluorescence lifetime imaging (FLIM) reveals protein microenvironment changes at the cell surface and immune synapse. This technique detects significant differences in green fluorescent protein (GFP) lifetimes, suggesting local refractive index variations.
Area of Science:
- Cell biology
- Biophysics
- Microscopy
Background:
- Green fluorescent protein (GFP) enables protein localization in live cells.
- Fluorescence lifetime imaging (FLIM) probes the protein's local microenvironment.
- Investigating protein behavior at the cell surface and immune synapse is crucial.
Purpose of the Study:
- Apply FLIM to GFP-tagged proteins at the cell surface and inhibitory natural killer (NK) cell immunological synapse (IS).
- Develop a quantitative analysis to determine statistical significance of FLIM heterogeneity.
- Explore potential local refractive index changes indicated by fluorescence lifetime variations.
Main Methods:
- Utilized fluorescence lifetime imaging (FLIM) on GFP-tagged proteins.
- Applied a novel quantitative analysis to fluorescence lifetime images.
- Compared fluorescence lifetimes of GFP-tagged proteins at the cell surface, in cells, and at the NK cell IS.
Main Results:
- Observed fluorescence lifetime variations of GFP-tagged proteins at the cell surface within the expected statistical range.
- Found shorter fluorescence lifetimes for GFP-tagged proteins in cells compared to recombinant GFP in solution.
- Detected a shortened fluorescence lifetime for GFP-tagged major histocompatibility complex class I protein at the inhibitory NK cell IS.
Conclusions:
- FLIM can detect statistically significant differences in fluorescence lifetimes.
- Observed lifetime variations suggest potential local refractive index changes in cellular microenvironments.
- FLIM offers a broadly applicable method for imaging discrete membrane environments of proteins.
Abstract:
Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.
