Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane

Eva Arnspang Christensen, J. Schwartzentruber, M. P. Clausen, P. W. Wiseman, B. C. Lagerholm

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.
Original languageEnglish
JournalPLOS ONE
Volume8
Issue number12
Pages (from-to)e78096
ISSN1932-6203
DOIs
Publication statusPublished - 2013

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Cell membranes
plasma membrane
Quantum Dots
Cell Membrane
quantum dots
Lipids
Molecules
Labeling
Semiconductor quantum dots
lipids
Spectroscopy
spectroscopy
Labels
Level measurement
methodology
Proteins
Macromolecules
proteins
Microscopes
microscopes

Cite this

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title = "Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane",
abstract = "The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.",
author = "Christensen, {Eva Arnspang} and J. Schwartzentruber and Clausen, {M. P.} and Wiseman, {P. W.} and Lagerholm, {B. C.}",
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Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane. / Christensen, Eva Arnspang; Schwartzentruber, J.; Clausen, M. P.; Wiseman, P. W.; Lagerholm, B. C.

In: PLOS ONE, Vol. 8, No. 12, 2013, p. e78096.

Research output: Contribution to journalJournal articleResearchpeer-review

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T1 - Bridging the gap between single molecule and ensemble methods for measuring lateral dynamics in the plasma membrane

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AU - Schwartzentruber, J.

AU - Clausen, M. P.

AU - Wiseman, P. W.

AU - Lagerholm, B. C.

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AB - The lateral dynamics of proteins and lipids in the mammalian plasma membrane are heterogeneous likely reflecting both a complex molecular organization and interactions with other macromolecules that reside outside the plane of the membrane. Several methods are commonly used for characterizing the lateral dynamics of lipids and proteins. These experimental and data analysis methods differ in equipment requirements, labeling complexities, and further oftentimes give different results. It would therefore be very convenient to have a single method that is flexible in the choice of fluorescent label and labeling densities from single molecules to ensemble measurements, that can be performed on a conventional wide-field microscope, and that is suitable for fast and accurate analysis. In this work we show that k-space image correlation spectroscopy (kICS) analysis, a technique which was originally developed for analyzing lateral dynamics in samples that are labeled at high densities, can also be used for fast and accurate analysis of single molecule density data of lipids and proteins labeled with quantum dots (QDs). We have further used kICS to investigate the effect of the label size and by comparing the results for a biotinylated lipid labeled at high densities with Atto647N-strepatvidin (sAv) or sparse densities with sAv-QDs. In this latter case, we see that the recovered diffusion rate is two-fold greater for the same lipid and in the same cell-type when labeled with Atto647N-sAv as compared to sAv-QDs. This data demonstrates that kICS can be used for analysis of single molecule data and furthermore can bridge between samples with a labeling densities ranging from single molecule to ensemble level measurements.

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