Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy
Henk-Jan van Manen 1, Paul Verkuijlen 2, Paul Wittendorp 1, Vinod Subramaniam 1, Timo van den Berg 3, Dirk Roos 3 and Cees Otto 1*
1 University of Twente
2 Sanquin research
3 Sanquin Research
* To whom correspondence should be addressed. E-mail: c.otto{at}utwente.nl.
Submitted on December 19, 2007
Revised on January 2, 2008
Accepted on 16 January 2008
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Abstract |
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We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ~1.38 and ~1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.
Key Words:
NADPH oxidase, fluorescence lifetime imaging, green fluorescent proteins, phagocytosis
