Descrizione del progetto
Microscopia elettronica per l’imaging 4D-STEM in biologia strutturale
La microscopia elettronica ha rivoluzionato le scienze fisiche e biologiche. La criomicroscopia elettronica consente di determinare le strutture proteiche 3D con una risoluzione fino a 1,0 Å; tuttavia, un contrasto insufficiente impedisce di visualizzare le strutture delle molecole coinvolte nelle malattie. La microscopia elettronica offre un contrasto ottimale per gli atomi luminosi, raggiungendo una notevole risoluzione pari a 0,2 Å mediante la trasformazione della STEM (scanning transmission electron microscopy, ovvero microscopia elettronica a trasmissione a scansione) da una tecnica di imaging 2D a una 4D, riuscendo a combinare in modo efficace l’immaginografia con la diffrazione. Il progetto 4D-BioSTEM, finanziato dal CER, mira a sviluppare metodologie e uno strumento di criomicroscopia elettronica per migliorare il contrasto e la risoluzione nell’imaging 4D-STEM di campioni biologici congelati. Il progetto si avvale di hardware specializzato, teoria, simulazione e algoritmi avanzati di ricostruzione delle immagini al fine dell’acquisizione di dati 4D sparsi, nonché dell’implementazione di tecniche avanzate per estrarre il massimo segnale da dati affetti da rumore ricavati mediante criomicroscopia elettronica a trasmissione a scansione 4D.
Obiettivo
Electron Microscopy (EM) has transformed research in the Life and Physical Sciences separately. Cryo-EM in Life Sciences allows the 3D structure determination of proteins down to 1.0 Å resolution in case they are large enough and present in high numbers in homogeneous states. For many molecules involved in diseases like neurodegeneration, however, structure determination is still severely hampered due to their insufficient contrast when imaged in vitreous ice or in their native cellular environment. To date, EM in the Physical Sciences generated utmost contrast for light atoms and established a resolution in the range of 0.2 Å limited only by thermal motion. This record was achieved by evolving scanning transmission EM (STEM) from a 2D to a 4D imaging technique combining imaging with diffraction. In this project, we will work out 4D-BioSTEM methodologies and develop a cryo-EM tool that maximizes contrast and resolution by bringing together EM groups from Life and Physical Sciences. 4D-STEM imaging of frozen biological specimens will be approached with unique and specialized hardware, theory and simulation, the development of microscope operation routines, and image reconstruction algorithms. In particular, we will acquire sparse 4D data using ultrafast detectors, employ methods for direct phasing (differential phase contrast, DPC) and establish advanced, so-called ptychographic, techniques to gain maximal signal from noisy 4D cryo-STEM data that are limited in their electron dose budget. We will make use of synergies between recently separated fields in order to make proteins smaller than 50 kDa amenable to structure elucidation. Furthermore, we will expand 4D-STEM to tomography, to obtain high-contrast 3D reconstructions from cellular samples, human brain tissue of neurodegenerative diseases, and vitrified organic energy materials. A new comprehensive structural imaging framework will be put forward and benchmarked as to its utility in Life and Physical Science applications of cryo-EM.
Campo scientifico
Parole chiave
Programma(i)
- HORIZON.1.1 - European Research Council (ERC) Main Programme
Argomento(i)
Meccanismo di finanziamento
HORIZON-ERC-SYG - HORIZON ERC Synergy GrantsIstituzione ospitante
52428 Julich
Germania