Home › Biochemistry › 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography
Biochemistry JoVE (Open Access) Citable · DOI

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

DOI: 10.3791/56567-v
What you'll learn
  • ✓ Apply serial-section electron tomography to visualize 3D mitochondrial ultrastructure
  • ✓ Prepare Drosophila flight muscle tissue using high-pressure freezing and freeze substitution
  • ✓ Generate and analyze serial EM sections for volumetric reconstruction
Protocol

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

Difficulty
advanced
Total time
~5–7 days per sample (including tissue preparation, freezing, sectioning, imaging, and 3D reconstruction)
Model organism
Drosophila melanogaster (indirect flight muscle)

Steps

1
Section Drosophila tissue with vibrating blade microtome

Prepare and section fresh or fixed Drosophila indirect flight muscle tissue using a vibrating blade microtome to generate thin sections suitable for electron microscopy.

▶ 00:37
2
Perform high-pressure freezing and freeze substitution

Apply high-pressure freezing (HPF) and freeze substitution (FS) protocols to preserve native tissue ultrastructure and prevent ice crystal artifacts during electron microscope sample preparation.

▶ 01:19
3
Generate serial sections for electron tomography

Systematically cut consecutive ultrathin serial sections from prepared tissue blocks and mount them on EM grids for imaging.

▶ 02:30
4
Acquire dual-axis electron tomography data

Collect high-resolution EM images of serial sections at two orthogonal tilt axes to improve 3D reconstruction accuracy and detail.

▶ 03:42
5
Visualize integrated cristae and matrix structure in 3D

Computationally reconstruct and analyze volumetric mitochondrial data to reveal cristae organization and matrix patterns in three dimensions.

▶ 05:09
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