Cell Biology JoVE (Open Access) Citable · DOI

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Name: Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis
Uploaded: 2026-07-01
Duration: 8 min 26 s
Description: Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

DOI: 10.3791/60461-v

What you'll learn

✓ Prepare silicon nitride membranes for cell culture and synchrotron imaging
✓ Execute plunge-freezing technique for cryopreservation of cultured cells
✓ Perform freeze-drying of frozen samples for room-temperature nano-analysis
✓ Interpret X-ray fluorescence data on intracellular elemental composition

Protocol

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

Difficulty

advanced

Total time

~3–5 days (including cell culture maturation, freezing, and freeze-drying cycles)

Model organism

Cell culture (HEK293 or equivalent mammalian cells)

Biosafety

BSL-1

Steps

Prepare silicon nitride membrane supports

Clean and treat silicon nitride membranes to render them suitable for cell adhesion and subsequent X-ray fluorescence analysis.

▶ 00:59

Seed cells onto prepared membranes

Plate cultured cells at appropriate density on silicon nitride membranes and allow attachment and growth prior to cryopreparation.

▶ 02:14

Plunge-freeze cell samples in cryogen

Rapidly immerse membrane-bound cells into liquid ethane or liquid nitrogen to achieve vitrification and preserve native cellular ultrastructure.

▶ 03:04

Freeze-dry plunge-frozen cell samples

Conduct lyophilization of frozen specimens to remove water while maintaining spatial distribution of intracellular elements for room-temperature X-ray analysis.

▶ 04:46

Acquire and interpret X-ray fluorescence images

Perform synchrotron cryogenic or room-temperature nano-XRF imaging on prepared samples to quantify intracellular elemental composition and distribution.

▶ 06:05

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