Biology subjects

Walla, P. J.

Publications and source records attributed to Walla, P. J..

3 recordsLinked to original sources

bioRxiv2020

Split-HaloTag Imaging Assay for in vivo 3D-Microscopy and Subdiffractional Analyses of Protein-Protein Interactions

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualisation of protein complex localisation. Due to its simple protocols, it has become one of the most frequently used methods. However, standard fluorescent proteins entail several drawbacks for sophisticated microscopy. With the HaloTag(R) system, these drawbacks can be overcome as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we established the Split-HaloTag(R) imaging assay in plants which is based on the reconstitution of a functional HaloTag(R) protein upon protein-protein interaction and subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using well-characterised interactions as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Split-GFP and Split-HaloTag(R) can complement one another as Split-HaloTag(R) represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag(R) imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interaction with specific microscopic techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy.

plant biology↗

bioRxiv2020

A device for exploring the full angular excitation space - Can more angular projections improve determination of a molecules 3D-orientation in the presence of noise?

In the past, different methods have been presented to determine the 3D orientation of single molecules in a microscopic set-up by excitation polarization modulation. Using linearly polarized illumination from different directions and thereby measuring different 2D projections enables reconstructing the full 3D orientation. Theoretically, two projections suffice for a full 3D orientation determination if the intensities are properly calibrated. If they are not, a third projection will enable unambiguous orientation measurements. The question arises if three projections already contain the maximum information on the 3D orientation when also considering the limited number of available photons and shot noise in an experiment, or if detecting more projections or even continuously changing the projection direction during a measurement provides more information with an identical number of available photons. To answer this principle question, we constructed a simple device allowing for exploring any projection direction available with a particular microscope objective and tested several different excitation modulation schemes using simulated as well as experimental single molecule data. We found that three different projections in fact already do provide the maximum information also for noisy data. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement. In fluorescence microscopy polarized illumination from different directions enables the determination of the 3D orientation of single molecules by combining the 2D information of different projection directions. Ambiguities that emerge when using only two projections can be eliminated using a third projection. In a systematic study we show that - also considering the limited number of available photons and shot noise in an experiment - three projection directions already contain the maximum information on the 3D orientation. Our results do not indicate a significant improvement in angular precision in comparison to three projections, both when increasing the number of projections and when modulating the projection direction and polarization simultaneously during the measurement.

biophysics↗

bioRxiv2020

Amplitude Analysis of Polarization Modulation Data and 3D-Polarization Demodulation (3D-SPoD)

Super-resolution optical fluctuation imaging (SOFI) is a technique that uses the amplitude of fluorescence correlation data for improved resolution of fluorescence images. Here, we explore if also the amplitude of superresolution by polarisation demodulation (SPoD) data can be used to gain additional information about the underlying structures. Highly organized experimental as well a simulated actin filament data demonstrate a principle information gain from this approach. In addition, we explored theoretically the benefits of analyzing the entire 3D-polarization information instead of only 2D-projections thereof. Due to fundamental principles, the probability of finding parallel orientations is approaching zero in 3D-SPoD in contrast to 2D-approaches. Using the modulation-amplitude based analysis we explored systematically simulated 3D-single molecules data (for which the true structures are known) under different conditions that are typically observed in experiments. We found that this approach can significantly improve the distinction, reconstruction and localization. In addition, these approaches are less sensitive to uncertainties in the knowledge about the true experimental point-spread-function (PSF) used for reconstruction compared to approaches using non-modulated data. Finally, they can effectively remove higher levels of non-modulated back-ground intensity.

biophysics↗