Microscopic morphologic observation of apoptosis

Summary

Observation of apoptosis by transmission electron microscopy can be used to: (1) clearly observe the changes of cell structure during different periods of apoptosis; (2) electron microscopy morphology is by far the most classical and reliable method to determine apoptosis, and is considered to be the gold standard for determining apoptosis.

Operation method

microscopic examination

Principle

Apoptosis occurs with certain morphological features, such as vesicle generation in the cytosol, cellular sequestration, nucleoplasmic condensation, chromatin condensation, DNA degradation, and eventual formation of many apoptotic vesicles in the cytosol, which are then phagocytosed by neighboring macrophages.

Materials and Instruments

Hela cells Jurkat cells
Distilled water 盼蓝
Optical microscope Inverted microscope Fluorescence microscope Confocal laser scanning microscope Transmission electron microscope

Move

Using a light microscope and appropriate staining methods, a series of morphological features that occur during apoptosis can be observed. Commonly used staining methods include Taipan Blue, Acridine Orange/Ethidium Bromide (AO/EB), Giemsa and HE. Typical apoptotic phenomena such as nuclear condensation, plasma condensation and apoptotic vesicles can be observed under the light microscope.

The electron microscopy morphological observation method is a more classical and reliable method, which is considered to be the gold standard for determining apoptosis. Under the electron microscope, apoptotic cells showed chromatin condensation, nuclear condensation, nuclear cleavage, cytoplasmic shrinkage and cytomembrane with foaming phenomenon and the appearance of apoptotic vesicles.

Caveat

The shortcomings of the morphological method are that it can only be qualitative, not effective quantitative, and the determination is inevitably subjective, so it is often used as a basis for other detection methods.

Common Problems

In apoptotic or necrotic cells, DNA breaks occur in the cell, small molecular weight DNA fragments increase, high molecular weight DNA decreases, and DNA fragments appear in the cytoplasm. However, in apoptotic cells, DNA breaks occur regularly between nucleosomes, and there are 180-200 DNA fragments, whereas in necrotic cells, DNA breaks are uncharacteristically disorganized, which makes it possible to determine the death of a group of cells and distinguish them from necrotic cells.

Sourced from Advances in Methods for the Detection of Apoptosis Journal of Biotechnology Bulletin.

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