Measurement of the fluorescence lifetime of GFP in high refractive index levitated droplets using FLIM.
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Author(s)
Type
Journal Article
Abstract
Green fluorescent protein (GFP) is a widely used fluorescent probe in the life sciences and biosciences due to its high quantum yield and extinction coefficient, and its ability to bind to biological systems of interest. This study measures the fluorescence lifetime of GFP in sucrose/water solutions of known molarity in order to determine the refractive index dependent lifetime of GFP. A range of refractive indices from 1.43-1.53 were probed by levitating micron sized droplets composed of water/sucrose/GFP in an optical trap under well-constrained conditions of relative humidity. This setup allows for the first reported measurements of the fluorescence lifetime of GFP at refractive indices greater than 1.46. The results obtained at refractive indices less than 1.46 show good agreement with previous studies. Further experiments that trapped droplets of deionised water containing GFP allowed the hygroscopic properties of GFP to be measured. GFP is found to be mildly hygroscopic by mass, but the high ratio of molecular masses of GFP to water (ca. 1500 : 1) signifies that water uptake is large on a per-mole basis. Hygroscopic properties are verified using brightfield microscope imaging, of GFP droplets at low and high relative humidity, by measuring the humidity dependent droplet size. In addition, this experiment allowed the refractive index of pure GFP to be estimated for the first time (1.72 ± 0.07). This work provides reference data for future experiments involving GFP, especially for those conducted in high refractive index media. The work also demonstrates that GFP can be used as a probe for aerosol studies, which require determination of the refractive index of the aerosol of any shape.
Date Issued
2020-06-15
Date Acceptance
2020-06-15
Citation
Physical Chemistry Chemical Physics, 2020, 22 (26), pp.14704-14711
ISSN
1463-9076
Publisher
Royal Society of Chemistry
Start Page
14704
End Page
14711
Journal / Book Title
Physical Chemistry Chemical Physics
Volume
22
Issue
26
Copyright Statement
© the Owner Societies 2020. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence (http://creativecommons.org/licenses/by/3.0/).
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/32573569
Subjects
02 Physical Sciences
03 Chemical Sciences
09 Engineering
Chemical Physics
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2020-06-15