High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Structural Biology

Background:

• Correlative light and electron microscopy (CLEM) integrates functional and structural imaging.
• Previous cryo-CLEM methods lacked nanometer-level precision for frozen-hydrated samples.
• Accurate correlation is crucial for understanding subcellular structures and processes.

Purpose of the Study:

• To develop a cryoCLEM workflow achieving nanometer-range precision for correlating fluorescence microscopy and cryo-electron tomography data.
• To enable precise localization of fluorescently tagged proteins within vitrified cells.
• To provide a method for estimating correlation accuracy for individual targets.

Main Methods:

• Vitrified whole cells were analyzed using correlative fluorescence cryo microscopy (cryoFM) and cryo-electron tomography (cryoET).
• Two independent electron-dense fluorescent markers were employed to enhance alignment precision.
• A step-by-step workflow from plunge-freezing to cryoET data acquisition was detailed.

Main Results:

• The developed cryoCLEM method achieves nanometer-range precision in correlating cryoFM and cryoET data.
• The workflow successfully located fluorescence-labeled adenovirus particles within cells.
• The method provides an estimate of correlation accuracy for each analyzed object.

Conclusions:

• This advanced cryoCLEM workflow significantly improves the precision of correlating fluorescence signals with high-resolution cryoET structures.
• The technique is applicable for precise localization of molecular targets within their native cellular context.
• This method opens new avenues for high-resolution structural and functional studies in cell biology.