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Experimental Parameters Leading to Optimal Bilayers for Total Internal Reflection Fluorescence Microscopy Visualization

Published online by Cambridge University Press:  23 February 2017

Elisabeth Mantil ,
Trinda Crippin ,
Anatoli Ianoul  and
Tyler J. Avis
Elisabeth Mantil
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Trinda Crippin
Affiliation:
Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Anatoli Ianoul
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
Tyler J. Avis*
Affiliation:
Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
*
*Corresponding author. tyler.avis@carleton.ca
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Abstract

Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.

Keywords

total internal reflection fluorescence microscopysmall unilamellar vesiclesextrusionlipid phase separationsupported lipid bilayers
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 23 , Issue 1 , February 2017 , pp. 97 - 112
Copyright
© Microscopy Society of America 2017 

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