Resource Capabilities

NCMIR capabilities merge leading-edge technologies, resources and expertise.  Our core capabilities are organized around four major categories:

Probes, Labeling Technologies, and Methods for Correlative, Multiscale Light, X-ray, and Electron Microscopy

NCMIR develops and applies next-generation molecular-genetic and chemical-labeling approaches to advance the application of high-resolution, correlated, multiscale and multimodal microscopy to biomedical research. With an expanded suite of methods, we aim to improve capabilities for determining the location and interactions of specific proteins in situ, a critical capability for projects where localization of molecular constituents in cell and tissue contexts will provide important new understanding.

CLEM Probes and Reagents

1.      MiniSOG for targeted multiscale protein labeling

2.      Split-MiniSOG for detecting and localizing protein-protein interactions

3.      APEX and APEX2 for protein localization in cells and tissues

4.      Split-APEX2 for proximity biotinylation for spatial proteomics and transcriptomics

5.      Split HRP for studying protein-protein interactions

6.      Click-EM for CLEM imaging of non-proteinaceous biomolecules

7.      ChromEMT for visualizing DNA

8.      Tetracysteine tags for multi-protein labeling (in cultured cells)

9.      Lanthenide-based labels for multicolor EM

10.    TimeSTAMP for tracking protein turnover in LM and EM

 

Specimen-preparation Methods for Multiscale – Multimodal Imaging

NCMIR offers methods for specimen preparation to rapidly broaden the application of advanced molecular probe technologies, particularly in support of multiscale, multimodal imaging. Unique specimen-preparation protocols and materials provide more suitable samples for examination when investigators use correlated microscopy in their research projects. These methods are meant to facilitate work on specific projects that now require the traversal of scales, which is becoming an increasing requirement of biomedical research projects that aim to advance understanding of molecular mechanisms underlying normal and abnormal cell and tissue biology. Our objectives are to create specimens suitable for wide-field, multi-color, and multi-resolution imaging using combinations of light microscopy (LM), X-ray microscopy (XRM), scanning EM (SEM), transmission EM (TEM), and scanning transmission EM (STEM) instruments.

Specimen Preparation Methods

1.      Fluorescence photo-oxidation for CLEM

2.      Fixation, embedding, and post-staining, including specialized XRM and SBEM preparations

3.      Plunge freezing, high pressure freezing and freeze substitution

4.      CryoChem fixation for correlated 3D CLEM

5.      Glow discharge and carbon coating of specimens

6.      Ultramicrotomy (and cryo-ultramicrotomy)

 

Instrumentation for Multiscale, Multimodal Imaging

NCMIR provides access to rare and highly specialized resources and instrumentation, specifically designed to obtain wider-field, higher-resolution data, including molecular localizations with multiple imaging modalities. Fo multple decades, the Center has developed, implemented, and refined the analytical capabilities of imaging instruments, such as NCMIR’s advanced high-throughput Intermediate-high Voltage TEM and SEM platforms. Our platforms facilitate projects aiming to differentiate complex structures, resolving ultrastructure and phenomena at higher resolution and on faster timescales.

Imaging Capabilities

1.      Widefield, confocal, multiphoton and intravital LM
2.      PALM/STORM super resolution LM
3.      Lattice light sheet microscopy (LLSM)
4.      X-ray Microscopy (XRM), microCT, and correlative LM, XRM, and EM
5.      SEM array tomography (widefield mosaicking and serial section imaging)
6.      Serial blockface SEM (SBEM) with focal charge compensation
7.      EM/STEM tomography (EMT)
8.      Energy filtered TEM (EFTEM), electron energy loss spectroscopy, and multicolor EM

 

Tools and Information Technology for Quantitative Data Refinement, Integration, and Analysis

Beyond high throughput data acquisition,  NCMIR provides software tools and infrastructure to refine, integrate, quantify, interpret, and add value to the increasingly complex multiscale image data derived through use of Resource technologies. We leverage scalable resources, including robustly connected Supercomputers and National research cluster and clouds.  We further leverages work supported by other grants to NCMIR Co-Is and collaborators, which bring to bear advanced cyberinfrastructure and new technologies for image processing and analysis (e.g.,  AI/deep learning for volume segmentation and enhancement). Where useful, we also incorporate the use of leading open-source and commercial-off-the-shelf (COTS) software tools. Our aim is better enable work with large multidimensional and multimodal data, employing methods and software tools that extend beyond what is commonly available.

Scalable Software Tools and Methods

1.      EM tomographic reconstruction

2.      Deep-learning based, automatic image segmentation

3.      Multiscale, multimodal data fusion and remapping/warping

4.      Ultra-widefield image mosaicking

5.      Z-stack alignment and normalization

6.      3D visualization, including volume and surface rendering and advanced meshing