Fluorescent and Electron-Dense Green Color Emitting Nanodiamonds for Single-Cell Correlative Microscopy.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Nanotechnology

Background:

• Correlative light and electron microscopy (CLEM) combines molecular and ultrastructural cell information.
• Existing fiducial markers for CLEM can be degraded by electron microscopy, limiting workflow.
• A need exists for robust fiducial markers that withstand electron microscopy and retain light microscopy properties.

Purpose of the Study:

• To evaluate green fluorescent and electron-dense nanodiamonds as fiducial landmarks for CLEM.
• To assess the stability of nanodiamond fluorescence under electron microscopy conditions.
• To demonstrate precise overlay of light and electron microscopy images using nanodiamonds.

Main Methods:

• Green fluorescent nanodiamonds were prepared and characterized.
• Nanodiamonds were applied as fiducial markers in CLEM experiments.
• Samples with nanodiamonds underwent electron microscopy, chemical treatments, and heavy metal staining.
• Fluorescence of nanodiamonds was assessed before and after electron microscopy and processing.

Main Results:

• Green fluorescent nanodiamonds were successfully used for precise correlation of light and electron microscopy images.
• Nanodiamonds demonstrated resistance to electron beam exposure.
• Nanodiamonds withstood harsh chemical treatments and heavy metal staining.
• Crucially, the fluorescent properties of the nanodiamonds remained intact after electron microscopy.

Conclusions:

• Green fluorescent nanodiamonds are versatile and robust fiducial markers for CLEM.
• These nanodiamonds overcome limitations of current fiducials by retaining fluorescence after electron microscopy.
• Their stability enables flexible CLEM workflows, allowing switching between imaging modalities.