Nanopipet delivery of individual molecules to cellular compartments for single molecule fluorescence tracking
Andreas Bruckbauer 1, Peter James 2, Dejian Zhou 1, Ji Won Yoon 1, David Excell 1, Yuri Korchev 3, Roy Jones 2 and David Klenerman 4*
1 University of Cambridge
2 Babraham Institute
3 Imperial College London
4 Cambridge University
* To whom correspondence should be addressed. E-mail: dk10012{at}cam.ac.uk.
Submitted on January 19, 2007
Revised on April 26, 2007
Accepted on 20 June 2007
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Abstract |
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We have developed a new method, using a nanopipet, 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: application of the probe to predefined regions on the membrane; release of only one or a few molecules onto the cell surface; combined with total internal reflection fluorescence (TIRF) microscopy there is very low background due to unbound molecules; the experiment can first be optimized and then repeated on the same cell. We validated the method by performing a SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin (WGA) 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 µm2/s in the postacrosomal region; this difference probably reflects different membrane structure. We also showed that we can analyze diffusional properties of different sub-regions of the cell membrane and probe for the presence of diffusion barriers. This new method should be straightforward to extend to other probes and cells and can be used as a new tool to investigate the cell membrane.
Key Words:
Plasma membrane, boar spermatozoa, scanning ion-conductance microscopy, single molecule fluorescence tracking, wheat germ agglutinin
