Home › Cell Biology › Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles
Cell Biology JoVE (Open Access) Citable · DOI

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

DOI: 10.3791/58109-v
What you'll learn
  • ✓ Purify Chlamydomonas centrioles using cell lysis and centrifugation techniques
  • ✓ Concentrate and immobilize centrioles on coverslips for imaging
  • ✓ Quantify isolated centrioles via immunofluorescence and super-resolution microscopy
  • ✓ Prepare centriole samples for single-particle averaging reconstruction
Protocol

Biopharma Insights We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

Difficulty
advanced
Total time
~4–6 hours per sample batch (purification through imaging)
Model organism
Chlamydomonas reinhardtii
Biosafety
BSL-1

Steps

1
Purify C. reinhardtii centrioles by cell lysis

Lyse Chlamydomonas cells and isolate centrioles using differential centrifugation to enrich the organelle fraction. This yields purified centrioles free of bulk cellular debris.

▶ 00:46
2
Concentrate centrioles onto coverslip center

Apply purified centriole suspension to coverslips and centrifuge to concentrate and immobilize them in a defined region suitable for high-resolution imaging.

▶ 05:15
3
Quantify isolated centrioles by immunofluorescence

Stain concentrated centrioles with fluorescent antibodies and image via super-resolution microscopy to count and verify centriole presence and orientation on coverslips.

▶ 07:06
4
Image centrioles for single-particle reconstruction

Acquire high-resolution fluorescence images of isolated centrioles in multiple orientations to enable single-particle averaging and 3D structural reconstruction.

▶ 08:47
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