Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy

Area of Science:

• Cellular ultrastructure and dynamics
• Correlative imaging techniques
• Cryo-electron microscopy (cryo-EM)
• Cryo-light microscopy (cryo-LM)

Background:

• Correlative cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) offer advantages for studying cellular dynamics and ultrastructure.
• Near-native sample environments and vitrification preserve cellular integrity.
• Current limitations include the high cost and complexity of specialized cryogenic stages.

Purpose of the Study:

• To demonstrate the assembly and use of an inexpensive, lab-fabricated cryogenic light microscopy stage.
• To enable direct correlative imaging of frozen hydrated samples using cryo-LM and cryo-EM.
• To make advanced correlative imaging accessible to laboratories with basic reflected light microscopes.

Main Methods:

• Fabrication of a low-cost cryogenic LM stage using readily available hardware store parts (under $40).
• Adaptation of standard 3-mm cryo-EM grids as specimen supports for correlative studies.
• Integration with reflected light microscopes equipped with long working distance air objectives.
• Utilized established protocols for sample vitrification, transfer, and handling.

Main Results:

• Successfully assembled and utilized an inexpensive cryogenic LM stage.
• Demonstrated the feasibility of correlative cryo-LM and cryo-EM imaging on the same frozen hydrated sample.
• Enabled direct correlation of data from single cells imaged with both techniques.

Conclusions:

• The developed cryogenic LM stage significantly lowers the barrier to entry for correlative cryo-LM and cryo-EM.
• This accessible technology empowers any laboratory with a reflected light microscope to perform advanced correlative imaging.
• Facilitates deeper understanding of cellular ultrastructure and dynamics through multi-modal imaging.