Home › Cell Biology › Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)
Cell Biology JoVE (Open Access) Citable · DOI

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

DOI: 10.3791/51469-v
What you'll learn
  • ✓ Apply fast 3D-SIM microscopy to visualize FtsZ protein dynamics in live bacteria
  • ✓ Prepare live bacterial samples for super-resolution imaging
  • ✓ Reconstruct and analyze 3D super-resolution image data of the Z ring
Protocol

Biopharma Insights Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Difficulty
advanced
Total time
~4–6 hours per experiment (sample preparation through image acquisition and reconstruction)
Model organism
Bacillus subtilis
Biosafety
BSL-1

Steps

1
Prepare live bacterial samples for imaging

Culture and prepare Bacillus subtilis expressing FtsZ-GFP fusion protein. Mount live cells on microscope slides with appropriate growth medium to maintain viability during imaging.

▶ 02:04
2
Calibrate and configure f3D-SIM microscope

Set up the OMX fast 3D-structured illumination microscope, including objective lens alignment, fluorescence detection channels, and illumination pattern parameters for bacterial imaging.

▶ 04:05
3
Acquire super-resolution image stacks

Capture 3D-SIM raw image data using structured illumination with multiple focal planes and phase shifts to resolve FtsZ-GFP localization and dynamics in live cells.

▶ 05:01
4
Reconstruct and validate 3D-SIM images

Process raw structured illumination data through computational reconstruction to generate super-resolution 3D images. Verify image quality and signal-to-noise metrics before analysis.

▶ 06:44
5
Generate 3D intensity plots of Z ring

Extract and visualize 3D intensity profiles from reconstructed images to quantify FtsZ distribution, bead-like organization, and spatial dynamics within the cytokinetic Z ring.

▶ 08:01
6
Interpret FtsZ localization and dynamics results

Analyze reconstructed 3D-SIM data to characterize FtsZ-GFP organization in the Z ring and quantify spatiotemporal dynamics of cytokinesis in live Bacillus subtilis.

▶ 09:58
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