My main research goal is to develop state-of-the-art single-particle cryogenic super-resolution fluorescence microscopy to uncover the intricate structures of soluble and membrane proteins in their native environments.
Additionally, our aim is to establish a streamlined workflow for freeze-preserved specimens enabling correlative structural biology studies using the two powerful microscopy approaches of cryogenic super-resolution light and electron microscopy.
Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa
Mahyar Dahmardeh, Houman Mirzaalian Dastjerdi, Hisham Mazal, Harald Köstler, Vahid Sandoghdar
Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise such that collection of more photons should extend its detection sensitivity to biomolecules of arbitrarily low mass. However, a number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the mass sensitivity limit by a factor of 4 to below 10 kDa. We implement this scheme both with a user-defined feature matrix and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal reflection mode. Our work opens the door to optical investigations of small traces of biomolecules and disease markers such as α-synuclein, chemokines and cytokines.<br><br>
Deciphering a hexameric protein complex with Angstrom optical resolution
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.
Insights into protein structure using cryogenic light microscopy
Hisham Mazal, Franz Wieser, Vahid Sandoghdar
Biochemical Society Transactions
(2023)
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Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.
Hisham Mazal studied Biotechnology Engineering (BSc) at ORT Braude Academic College of Engineering (Israel) from 2010 to 2013 and Chemical and Biological Physics (MSc) at Weizmann Institute of Science (Israel) from 2013 to 2015 as an undergraduate student. From 2016 to 2020 he continued at Weizmann Institute for his PhD thesis on “Single-molecule protein dynamics: From ligand binding effects on folding to function-related motions” and as a postdoc. In June 2020 Hisham Mazal joined the group of Prof. Vahid Sandoghdar at MPL as a postdoc.