Hot-band absorption and anti-Stokes emission properties of an organic fluorescent dye, Alexa Fluor 568, were characterized and compared with those of Rhodamine 101. The comparison of the properties (e.g., quantum efficiency, spectral distribution, thermal properties, and fluorescence lifetime) between the two dyes confirms that both dyes undergo the same process when excited in the red spectral region. Possible undesirable crosstalk effects and applications in dSTORM microscopy were demonstrated and discussed.
Gajdos, T., Hopp, B. & Erdélyi, M. Hot-Band Anti-Stokes Fluorescence Properties of Alexa Fluor 568. J Fluoresc (2020). doi: 10.1007/s10895-020-02496-0
Sarcomeres are extremely highly ordered macromolecular assemblies where structural organization is intimately linked to their functionality as contractile units. Although the structural basis of actin and Myosin interaction is revealed at a quasiatomic resolution, much less is known about the molecular organization of the I-band and H-zone. We report the development of a powerful nanoscopic approach, combined with a structure-averaging algorithm, that allowed us to determine the position of 27 sarcomeric proteins in Drosophila melanogaster flight muscles with a quasimolecular, ∼5- to 10-nm localization precision. With this protein localization atlas and template-based protein structure modeling, we have assembled refined I-band and H-zone models with unparalleled scope and resolution. In addition, we found that actin regulatory proteins of the H-zone are organized into two distinct layers, suggesting that the major place of thin filament assembly is an M-line–centered narrow domain where short actin oligomers can form and subsequently anneal to the pointed end.
Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes – Szilárd Szikora, Tamás Gajdos, Tibor Novák, Dávid Farkas, István Földi, Peter Lenart, Miklós Erdélyi, József Mihály
… based on our new analysis method, we were able to show the number of nucleosomes in each nanofocus that could allow us to define the possible chromatin structure and the nucleosome density around the break sites. This method is one of the first demonstration of a single-cell based quantitative measurement of a discrete repair focus, which could provide new opportunities to categorize spatial organization of nanofoci by parametric determination of topological similarity.
Super-resolution localization microscopy provides a powerful tool to study biochemical mechanisms at single molecule level. Although the lateral position of the fluorescent dye molecules can be determined routinely with high precision, measurement of other modalities such as 3D and multicolor without the degradation of the original super-resolved image is still in the focus. In this paper a dual-objective multimodal single molecule localization microscopy (SMLM) technique has been developed, optimized and tested. The proposed optical arrangement can be implemented onto a conventional inverted microscope without serious system modification. Read more
Replaced Matlab’s iteration to a custom one for increased reliability when calculating scalar or vectorial PSFs.
Modification to the focal position determination when calculating scalar or vectorial PSFs. Results only slightly different focal position than the previous algorithm, in normal cases (~22 nm difference with the default settings).
Fixed a bug when calculating scalar or vectorial PSFs (not sure whether a new Matlab version caused it or it was originally in the code). The PSFs shapes and peaks were only marginally affected.
Made a workaround for a bug causing higher blinking event brightness when simulating with very low “Sample depth” value.
Added “Synaptosomes” option to the random “Vesicles” pattern.
Parameters files exported with previous TestSTORM versions (2.0.x) and containing “Rods”, “Vesicles” or “Octagons” patterns can not be imported any longer starting this version.
Added a tool with which the generated PSF can be depicted.
Other clean-ups and modification in the code which should not affect the behaviour of the program.
Optimization of sample, imaging and data processing parameters is an essential task in localization based super-resolution microscopy, where the final image quality strongly depends on the imaging of single isolated fluorescent molecules. A computational solution that uses a simulator software for the generation of test data stacks was proposed, developed and tested. The implemented advanced physical models such as scalar and vector based point spread functions, polarization sensitive detection, drift, spectral crosstalk, structured background etc., made the simulation results more realistic and helped us interpret the final super-resolved images and distinguish between real structures and imaging artefacts.
A practical method has been presented for polarization sensitive localization based super-resolution microscopy using a birefringent dual wedge. The measurement of the polarization degree at single molecule level can reveal the chemical and physical properties of the local environment of the fluorescent dye molecule and can hence provide information about the sub-diffraction sized structure of biological samples. Polarization sensitive STORM imaging of the F-actins proved correlation between the orientation of fluorescent dipoles and the axis of the fibril.
A figure was selected for the cover page from the article Manetsberger, J., Manton, J. D., Erdelyi, M. J., Lin, H., Rees, D., Christie, G., & Rees, E. J. (2015). Ellipsoid Localization Microscopy Infers the Size and Order of Protein Layers in Bacillus Spore Coats. Biophysical Journal, 109(10), 2058-2066.