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Preparation of Samples for Electron Microscopy01:20

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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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Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
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Saline dry fixation for improved cell composition analysis using Raman spectroscopy.

Shreyas Rangan1,2, Riley Wong1, H Georg Schulze3

  • 1Michael Smith Laboratories, The University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada. turner@msl.ubc.ca.

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Dry-fixation with saline preserves more cellular information in Raman spectroscopy compared to traditional chemical fixatives like formaldehyde or methanol. This method offers stable, reliable cellular analysis for collaborative research.

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Area of Science:

  • Biophotonics
  • Cellular Imaging
  • Spectroscopy

Background:

  • Raman spectroscopy offers label-free cellular composition analysis.
  • Fixed cell analysis is valuable for collaborative and long-term studies.
  • Chemical fixatives can alter cellular biomolecules, affecting Raman spectra.

Purpose of the Study:

  • To compare the efficacy of dry-fixation with saline versus chemical fixatives (formaldehyde, methanol) for cellular Raman spectroscopy.
  • To evaluate the stability of dry-fixed cells over time.
  • To compare dry-fixed and live cell spectra.

Main Methods:

  • Cell samples were fixed using dry-fixation with saline, formaldehyde, and methanol.
  • Raman spectroscopy was performed on fixed and live cells.
  • Dry-fixed cells were assessed for stability over 5 months.

Main Results:

  • Dry-fixed cell spectra retained more cellular information than formaldehyde or methanol-fixed cells.
  • Dry-fixed cells remained stable for at least 5 months.
  • Rehydrating dry-fixed samples recovered spectral features observed in live cells, indicating hydration effects.

Conclusions:

  • Dry-fixation with unbuffered saline is a superior method for fixed cell Raman spectroscopy compared to formaldehyde or methanol fixation.
  • This method preserves cellular information and offers stability for extended analysis.
  • Understanding hydration effects is crucial for interpreting dry-fixed cell Raman spectra.