Imaging the environment of green fluorescent protein.
An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.
Suhling
Klaus
K
Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College of Science, Technology and Medicine, London SW7 1AZ, United Kingdom.
Siegel
Jan
J
Phillips
David
D
French
Paul M W
PM
Lévêque-Fort
Sandrine
S
Webb
Stephen E D
SE
Davis
Daniel M
DM
D005453:Fluorescence; D005632:Fructose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins; D007834:Lasers; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects; D008856:Microscopy, Fluorescence / Q000379:methods; D011092:Polyethylene Glycols / Q000737:chemistry; D012031:Refractometry / Q000379:methods*; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005632:Fructose; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D005947:Glucose; D005990:Glycerol