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 microm(2)/s in the acrosomal region and 0.10 +/- 0.02 microm(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.

On-Demand Delivery of Single DNA Molecules Using Nanopipets

“…Due to their high-aspect ratio geometry and exceptionally sharp tips, nanopipettes offer an important advantage over conventional nanopores as they can be easily adapted for use in single cell interrogation 15,16 and as a macroscopic delivery vehicle for intracellular injection. [17][18][19][20][21][22] . To date, delivery of molecules has so far only been quantified in close proximity to the tip by using fluorescence spectroscopy.…”

Multifunctional scanning ion conductance microscopy

“…Additionally fluorescence measurements have been very valuable in following the dynamics of delivery from nanopipettes and the behaviour of the fluorescent moieties while in a nanopipette [75,152]. These studies showed that it was possible to deliver a single fluorescent molecule to different subcellular regions of a boar spermatozoon and track the diffusion therein [152]. SICM has also been combined with scanning near field optical microscopy [153].…”

Imaging the cell surface and its organization down to the level of single molecules

“…This enables controlled delivery of molecules onto a surface at defined positions, and by controlling the concentration of solution in the pipette and the applied voltage and dose time, delivery of individual molecules is possible [17 -20]. This has been used to study the diffusion of molecules over the surface of a boar sperm and probe the nature of the barrier between different macrodomains on the surface, when combined with single-molecule tracking, and is discussed in more detail below [21]. We have also trapped fluorophores in the tip of the pipette due to the force generated by dielectrophoresis (induced dipole forces) [22].…”