Cryo-electron microscopy has revolutionized the field of structural biology, primarily for macromolecular structure but also for cells and tissue sections—achieving resolutions at the limit of physical optics. While wide-field tra...
Cryo-electron microscopy has revolutionized the field of structural biology, primarily for macromolecular structure but also for cells and tissue sections—achieving resolutions at the limit of physical optics. While wide-field transmission EM (TEM) with phase contrast by defocus is the most commonly used modality in biology, the alternative, scanning transmission EM (STEM), has emerged as the mode of choice for atomic resolution in materials science. Seeking to endow biology with the benefits of STEM, our lab established STEM for cryo-tomography of biological cells and demonstrated its advantages for thick specimens and compositional contrast. We now seek to extend cryo-STEM to high-resolution, with an emphasis on tomography, by means of coherent detection (Obj1). This will be achieved by the method of integrated differential phase contrast (iDPC) using a segmented detector, from which we obtain simultaneously phase and depth contrast in a single scan.
The major expected benefits are: 1) minimization of image aberration, especially defocus with its associated complications for image interpretation, 2) reduction of beam-induced radiation damage by means of flexible scan and sampling patterns, and 3) improved reconstruction for tomography based on tailored data acquisition. We will validate the new methods for single particle analysis on standard macromolecular substrates and compare them to current state-of-the-art methods. Further, we will apply the new developments in 3D imaging to explore novel large-scale structures in chromatin we observed recently by whole-cell cryo-STEM tomography using current, low-resolution methods (Obj2). Labelling with halogenated nucleotides will reveal sites of active transcription or DNA synthesis.
The proposed approaches’ expected broad applicability and STEM’s unrealized potential for hardware simplicity should together ensure the wide adoption of cryo-STEM methods in biology, accelerated by our dissemination efforts (Obj3).ver más
02-11-2024:
Generación Fotovolt...
Se ha cerrado la línea de ayuda pública: Subvenciones destinadas al fomento de la generación fotovoltaica en espacios antropizados en Canarias, 2024
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