01. April 2025, 16:00 until 17:00

Anna Gaugutz, TU Wien, IAP, FB Biophysik

Seminar

3D Localisation Microscopy and Single Molecule Tracking of DNA Nanostructures and Cell Membrane Proteins Using Defocused Imaging

At the interface of interacting cells, the 3D topography of the plasma membranes influences biomolecular interactions, for example by sterically hindering the diffusion of membrane proteins. A prominent example is the exclusion of the phosphatase CD45 with its large extracellular domain from the close contact zone during T cell activation . To investigate these exclusion effects, it is essential to track individual membrane proteins in 3D. While standard single molecule localisation microscopy (SMLM) offers high localisation precision in lateral dimensions, its performance in the axial z- direction is limited. Here we describe a new version of SMLM based on defocused imaging which allows tracking of single proteins in 3D and achieves a resolution of 10 nm along the axial direction. The method is validated by tracking two DNA nanoprobes (DNA-NPs) on supported lipid bilayers. DNA-NPs are custom designed using DNA nanotechnology and are labeled with two fluorophores separated by a distance of 11 nm in the z-axis. Using 3D SMLM, we determine a lateral diffusion coefficient of D ≈ 0.3 μm2/s and a relative height difference of Δz = 10.43 ± 1.16 nm. Furthermore, we apply our method to track the 3D diffusion of T cell receptors on the cell surface and thereby reveal the morphology of the immune synapse and the microvilli. Our findings lay the foundation for high-resolution 3D imaging and enable the analysis of protein dynamics on the membrane of living cells.

Calendar entry

Event location

SEM.R. DB gelb 05 B
1040 Wien
Wiedner Hauptstraße 8-10/E134

 

Organiser

IAP
Manuela Marik
marik@iap.tuwien.ac.at

 

Public

Yes

 

Entrance fee

No

 

Registration required

No