Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment by 3D-Structured Illumination Microscopy (3D-SIM) (CAT#: STEM-MIT-0411-LJX)

Introduction

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This service provides a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM).

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Principle Applications Procedure Materials Notes Related Products

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:
Rat hippocampal neurons

Notes

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

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