Quantitative accounting of dye leakage and photobleaching in single lipid vesicle measurements: Implications for biomacromolecular interaction analysis.
Colloids Surf B Biointerfaces
; 182: 110338, 2019 Oct 01.
Article
en En
| MEDLINE
| ID: mdl-31301580
ABSTRACT
Highly parallel measurements on single, tethered lipid vesicles enable real-time monitoring of dynamic membrane interactions of relevance to medical, pharmaceutical, and biotechnological applications. Monitoring the time-dependent release of entrapped fluorescent dyes is a popular measurement approach, although it is often challenging to accurately extract quantitative biochemical parameters. Key issues include dye leakage and imaging-related photobleaching, and corrective measures are needed. Herein, we present an extended analytical framework to collect and interpret time-lapsed fluorescence microscopy imaging data, and demonstrate its utility for tracking membrane-peptide interactions. Our approach is focused on improving platform design and data analysis. First, we identified suitable membrane compositions to minimize dye leakage while enhancing the biomimetic character of lipid vesicles. Second, a data normalization procedure was developed to correct for experimental artifacts, namely dye leakage and photobleaching, and hence improve measurement accuracy. This analytical procedure was applied to experimentally determine the rate of peptide-induced pore formation in single lipid vesicles, and there was up to a nearly three-fold decrease in the measured rate, as compared to uncorrected data. Taken together, the results present a broadly applicable analytical framework to account for experimental artifacts and improve measurement accuracy in highly parallel, single lipid vesicle arrays.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Péptidos
/
Fosfatidiletanolaminas
/
Rodaminas
/
Liposomas Unilamelares
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Fluoresceínas
/
Colorantes Fluorescentes
/
Membrana Dobles de Lípidos
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Colloids Surf B Biointerfaces
Asunto de la revista:
QUIMICA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Singapur