Enhancing FIB-SEM with katana microtome. A Correlative EM Workflow with FIB, SBF and Light Microscopy

Introduction

Through BBSRC Facility Access Funding for Bioscience Partnerships, a collaboration between the UK Health Security Agency (UKHSA), Central Laser Facility (CLF), and ConnectomX has developed an innovative workflow for efficient volume imaging of thick tissue samples. This workflow employs correlative light, serial block-face (SBF), and focused ion beam (FIB) microscopy to investigate the impact of inhaled particles on the blood-brain barrier (BBB) in animal models, with a focus on dementia-related brain health.

Background

Air pollution is a recognised risk factor for dementia, contributing to approximately 40% of cases through modifiable factors (Lancet Commission, 2020). Inhaled particles may translocate to the brain via the olfactory nerve or bloodstream, potentially disrupting the BBB and triggering neuroinflammation. This project utilised scanning electron microscopy to examine particle distribution and BBB integrity, particularly in the hippocampus—a region associated with memory loss and Alzheimer’s disease.

Workflow Overview

The workflow consists of the following steps:

• Animal Exposure and Tissue Preparation: Animals are exposed to particles, and brain tissue is sectioned into 200 µm thick slices.

• Fiducial Marking: A two-photon microscope laser creates fiducial markers (termed "branding") to identify regions of interest (ROIs).

• Sample Processing: Samples are stained with heavy metals and embedded in resin.

• SBF-SEM Imaging: A Zeiss Crossbeam SEM, equipped with a katana microtome, serially cuts and images the sample to locate the branding.

• Automated Detection: An algorithm detects the branding, halting imaging once the ROI is identified.

• FIB-SEM Imaging: High-magnification imaging of the ROI is performed using FIB-SEM.

Key Findings

• Enhanced Efficiency: The katana microtome improved correlative light and electron microscopy (CLEM) by enabling efficient SBF-SEM imaging of thick brain sections.

  
  

• BBB Damage: Imaging revealed BBB disruptions, including edema and irregular endothelial structures, with dark agglomerates likely representing translocated particles.

  
  

• Neurological Impact: The workflow provided evidence that inhaled particles compromise BBB integrity, potentially contributing to neurological health decline.