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To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...
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Sample Preparation Protocol for Laboratory Cryo-Soft X-Ray Microscopy for Studying Cellular Nanoparticle Uptake.

Komang G Y Arsana1, Martin Svenda1, Hans M Hertz1

  • 1Biomedical and X-Ray Physics, Department of Applied Physics, KTH Royal Institute of Technology, 10691 Stockholm, Sweden.

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|February 26, 2025
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Summary

This study presents a sample preparation protocol for laboratory soft X-ray microscopy (LSXM) to visualize cellular nanoparticle uptake. Optimized vitrification ensures thin, high-contrast samples for improved imaging of biological structures.

Keywords:
X-ray microscopycell imagingcryofixationnanoparticlesample preparationwater window

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Area of Science:

  • Biomedical Imaging
  • Cellular Biology
  • Nanotechnology

Background:

  • Soft X-ray microscopy (SXM) offers high-resolution imaging of biological samples in near-native states.
  • Laboratory soft X-ray microscopes (LSXMs) aim to bridge the resolution gap between light and electron microscopy.
  • LSXM contrast can be limited by lower X-ray source brightness compared to synchrotrons, necessitating optimized sample preparation.

Purpose of the Study:

  • To develop and detail a sample preparation protocol for LSXM focused on cellular nanoparticle uptake.
  • To optimize sample preparation for LSXM to overcome contrast limitations associated with laboratory-based X-ray sources.
  • To demonstrate the protocol's efficacy in visualizing nanoparticle interactions within different cell types.

Main Methods:

  • Development of optimized parameters for manual plunge-freezing and automated vitrification techniques.
  • Ensuring rapid transition of biological samples into a vitrified state with controlled ice thickness (5-10 μm).
  • Application of the protocol to murine macrophages and acanthamoeba for nanoparticle uptake studies.

Main Results:

  • The protocol successfully preserved cellular structures during rapid freezing.
  • Controlled ice thickness facilitated optimal X-ray transmission, enhancing image contrast.
  • Effective visualization of nanoparticle uptake was achieved in both murine macrophages and acanthamoeba.

Conclusions:

  • Optimized sample preparation, particularly controlling ice thickness, is crucial for high-quality SXM imaging using LSXMs.
  • This protocol enables detailed investigation of cellular nanoparticle interactions with improved contrast and resolution.
  • The developed method enhances the utility of LSXM for biomedical research, particularly in nanomedicine and toxicology.