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Monitoring autophagy using super-resolution structured illumination microscopy (CAT#: STEM-MIT-0403-LJX)

Introduction

Autophagy is a major protein degradation pathway responsible for the removal of primarily long-lived and misfolded proteins, contributing to cellular homeostasis. Autophagy dysfunction has been associated with the onset of various human pathologies. Visualizing key proteins that govern autophagy pathway activity, the molecular machinery and cargo is essential to elucidate roles and mechanisms of autophagy function. Although multiple fluorescence-based microscopy approaches exist to assess autophagy, the limit of resolution associated with light microscopy makes precise intracellular protein localization, interaction and molecular distribution challenging. The service describes a detailed protocol for both super-resolution structured illumination microscopy (SR-SIM) as well as direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of key proteins associated with the autophagy molecular machinery and cargo.




Principle

The structured illumination microscopy (SIM) applies a pattern lighting field (different from the traditional wide-field lighting) to the samples to improve the spatial resolution of the optical microscope and has advantages for the observation of living cells. In this method, the spatial frequency of the illumination pattern is mixed with the spatial frequency of the sample feature, converting the high frequency feature into a lower frequency detectable by the microscope. The periodic lighting pattern (Moire fringes, Moire fringes) is generated by the interference of multiple light sources in the axial (Z), lateral (X-Y) or both directions, and the high-resolution image is reconstructed based on the acquisition of multiple illumination images in different phases and directions. Since the illumination mode itself is also limited by optical diffraction, SIM can only double the spatial resolution by combining two information sources with limited diffraction, achieving resolutions of 100 nm and 300 nm in the X-Y and Z-axis directions, respectively.

Applications

Applied to the research of cell physiology, cell dynamics and other subcellular level

Procedure

1. Sampling
2. Preparation of slices
3. Staining (Select according to the specific experimental situation)
4. Observation

Materials

• Sample Type:
Autophagy cells

Notes

Operate in strict accordance with the operating procedures, and shall not arbitrarily change the operating procedures
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