Insight
is our reward
Search

Publications in Microscopy by NOMIS researchers

Whole-body cellular mapping in mouse using standard IgG antibodies

NOMIS Researcher(s)

Published in

July 10, 2023

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-β-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php . © 2023, The Author(s).

Research field(s)
Applied Sciences, Enabling & Strategic Technologies, Biotechnology

DOI
10.1038/s41587-023-01846-0

Whole-mouse clearing and imaging at the cellular level with vDISCO

NOMIS Researcher(s)

Published in

January 25, 2023

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies. © 2023, Springer Nature Limited.

Research field(s)
Applied Sciences, Enabling & Strategic Technologies, Bioinformatics

DOI
10.1038/s41596-022-00788-2

Correlative all-optical quantification of mass density and mechanics of sub-cellular compartments with fluorescence specificity

NOMIS Researcher(s)

Published in

January 1, 2022

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.7554/eLife.68490

Fully automated, sequential focused ion beam milling for cryo-electron tomography

NOMIS Researcher(s)

Published in

March 1, 2020

Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here, we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.

Research field(s)
Health Sciences, Biomedical Research, Developmental Biology

DOI
10.7554/eLife.52286

Improved applicability and robustness of fast cryo-electron tomography data acquisition

NOMIS Researcher(s)

Published in

November 1, 2019

The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared to single particle approaches. A recent study reported significant acceleration of data acquisition by a fast-incremental single-exposure (FISE) tilt series scheme. Here we improved the method and evaluated its efficiency and performance. We show that (1) FISE combined with the latest generation of direct electron detectors speeds up collection considerably, (2) previous generation (pre-2017) double-tilt axis Titan Krios holders are also suitable for FISE data acquisition, (3) x, y and z-specimen shifts can be compensated for, and (4) FISE tilt series data can generate averages of sub-nanometer resolution. These advances will allow for a widespread adoption of cryoET for high-throughput in situ studies and high-resolution structure determination across different biological research disciplines.

Research field(s)
Health Sciences, Biomedical Research, Biophysics

DOI
10.1016/j.jsb.2019.08.006

  • NOMIS Foundation 2025
  • 2025 NOMIS Foundation