Biology subjects

D'Imprima, E.

Publications and source records attributed to D'Imprima, E..

5 recordsLinked to original sources

bioRxiv2026

Workflow and instrumentation for multiscale correlative imaging in voluminous cryogenic specimens

Cryo-electron tomography (cryo-ET) enables molecular-resolution imaging of intracellular environments, but is constrained by the need to generate electron-transparent sections of the biological specimen and thus a limited imaging volume. To alleviate such limitations, we present a multiscale correlative workflow that links cryo-confocal fluorescence microscopy (cryo-FLM), cryo-focused ion beam-scanning electron microscopy (cryo-FIB-SEM) volume imaging (cryo-vEM), and cryo-FIB serial lamellae lift-out for cryo-ET in high-pressure frozen voluminous specimens. An integral part of this workflow is a sample holder that preserves specimen orientation across instruments, reduces manual handling, and overcomes stage-tilt limitations in cryo-FIB-SEM hardware. Applied to vitrified colorectal cancer organoids, we demonstrate that this workflow enables fluorescence-guided site preparation for cryo-vEM, which provides biological context beyond the limited field of view of cryo-ET, and targeted lift-out for fabrication of multiple lamellae from defined regions of interest. The reconstructed tomograms reveal subcellular architecture and cell-cell interfaces. This workflow establishes a modular methodological framework for continuum-resolution cryogenic imaging of complex 3D biological specimens.

cell biology↗

bioRxiv2024

Amortized template-matching of molecular conformations from cryo-electron microscopy images using simulation-based inference

AO_SCPLOWBSTRACTC_SCPLOWBiomolecules undergo conformational changes to perform their function. Cryo-electron microscopy (cryo-EM) can capture snapshots of biomolecules in various conformations. However, these images are noisy and display the molecule in unknown orientations, making it difficult to separate conformational differences from differences due to noise or projection directions. Here, we introduce cryo-EM simulation-based inference (cryoSBI) to infer the conformations of biomolecules and the uncertainties associated with the inference from individual cryo-EM images. CryoSBI builds on simulation-based inference, a combination of physics-based simulations and probabilistic deep learning, allowing us to use Bayesian inference even when likelihoods are too expensive to calculate. We begin with an ensemble of conformations, which can be templates from molecular simulations or modelling, and use them as structural hypotheses. We train a neural network approximating the Bayesian posterior using simulated images from these templates, and then use it to accurately infer the conformations of biomolecules from experimental images. Training is only done once, and after that, it takes just a few milliseconds to make inference on an image, making cryoSBI suitable for arbitrarily large datasets. CryoSBI eliminates the need to estimate particle pose and imaging parameters, significantly enhancing the computational speed in comparison to explicit likelihood methods. We illustrate and benchmark cryoSBI on synthetic data and showcase its promise on experimental single-particle cryo-EM data.

biophysics↗

bioRxiv2021

Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before. SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

cell biology↗

bioRxiv2019

The resolution revolution in cryoEM requires new sample preparation procedures: A rapid pipeline to high resolution maps of yeast FAS

Single-particle electron cryo-microscopy (cryoEM) has undergone a \"resolution revolution\" that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 [A] map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

biochemistry↗

bioRxiv2018

The deadly touch: protein denaturation at the water-air interface and how to prevent it

Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation of the protein. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions are those facing the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

biophysics↗