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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 4, Iss. 8 — Aug. 1, 2013
  • pp: 1390–1400

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Ali Vaziri Gohar, Ruofan Cao, Patrick Jenkins, Wenyan Li, Jessica P. Houston, and Kevin D. Houston  »View Author Affiliations


Biomedical Optics Express, Vol. 4, Issue 8, pp. 1390-1400 (2013)
http://dx.doi.org/10.1364/BOE.4.001390


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Abstract

Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

© 2013 OSA

OCIS Codes
(170.1530) Medical optics and biotechnology : Cell analysis
(170.6920) Medical optics and biotechnology : Time-resolved imaging
(260.2510) Physical optics : Fluorescence
(140.3518) Lasers and laser optics : Lasers, frequency modulated

ToC Category:
Cell Studies

History
Original Manuscript: June 10, 2013
Revised Manuscript: July 11, 2013
Manuscript Accepted: July 15, 2013
Published: July 19, 2013

Citation
Ali Vaziri Gohar, Ruofan Cao, Patrick Jenkins, Wenyan Li, Jessica P. Houston, and Kevin D. Houston, "Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry," Biomed. Opt. Express 4, 1390-1400 (2013)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-4-8-1390


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