Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking

Andreas Bruckbauer, Peter James, Dejian Zhou, Ji Won Yoon, David Excell, Yuri Korchev, Roy Jones, David Klenerman

Research output: Contribution to journalArticle

77 Citations (Scopus)

Abstract

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm 2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.

Original languageEnglish
Pages (from-to)3120-3131
Number of pages12
JournalBiophysical Journal
Volume93
Issue number9
DOIs
Publication statusPublished - 2007 Nov 1
Externally publishedYes

Fingerprint

Fluorescence
Cell Membrane
Membranes
Membrane Glycoproteins
Fluorescence Microscopy
Triticum
Spermatozoa

ASJC Scopus subject areas

  • Biophysics

Cite this

Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking. / Bruckbauer, Andreas; James, Peter; Zhou, Dejian; Yoon, Ji Won; Excell, David; Korchev, Yuri; Jones, Roy; Klenerman, David.

In: Biophysical Journal, Vol. 93, No. 9, 01.11.2007, p. 3120-3131.

Research output: Contribution to journalArticle

Bruckbauer, A, James, P, Zhou, D, Yoon, JW, Excell, D, Korchev, Y, Jones, R & Klenerman, D 2007, 'Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking', Biophysical Journal, vol. 93, no. 9, pp. 3120-3131. https://doi.org/10.1529/biophysj.107.104737
Bruckbauer, Andreas ; James, Peter ; Zhou, Dejian ; Yoon, Ji Won ; Excell, David ; Korchev, Yuri ; Jones, Roy ; Klenerman, David. / Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking. In: Biophysical Journal. 2007 ; Vol. 93, No. 9. pp. 3120-3131.
@article{fb272d3ee1404da49afc2805bc4bd8bc,
title = "Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking",
abstract = "We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm 2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.",
author = "Andreas Bruckbauer and Peter James and Dejian Zhou and Yoon, {Ji Won} and David Excell and Yuri Korchev and Roy Jones and David Klenerman",
year = "2007",
month = "11",
day = "1",
doi = "10.1529/biophysj.107.104737",
language = "English",
volume = "93",
pages = "3120--3131",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "9",

}

TY - JOUR

T1 - Nanopipette delivery of individual molecules to cellular compartments for single-molecule fluorescence tracking

AU - Bruckbauer, Andreas

AU - James, Peter

AU - Zhou, Dejian

AU - Yoon, Ji Won

AU - Excell, David

AU - Korchev, Yuri

AU - Jones, Roy

AU - Klenerman, David

PY - 2007/11/1

Y1 - 2007/11/1

N2 - We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm 2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.

AB - We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm2/s in the acrosomal region and 0.10 ± 0.02 μm 2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.

UR - http://www.scopus.com/inward/record.url?scp=34447282639&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34447282639&partnerID=8YFLogxK

U2 - 10.1529/biophysj.107.104737

DO - 10.1529/biophysj.107.104737

M3 - Article

VL - 93

SP - 3120

EP - 3131

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 9

ER -