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Cytology · Year 1 · Medical University of Sofia

09

Cell fractionation. Histoautoradiography

Free notes for topic 09 of the Cytology syllabus, open without an account. Written by a senior student against the syllabus question and checked line by line by a second student before publishing. How content is made

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Summary

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Two ways of finding out where something is in a cell, and one of finding out what shape it is.

Cell fractionation takes the cell apart and sorts the pieces by size. It has two stages: homogenisation, which breaks the cells open and gives a homogenate, and fractionation by centrifugation, which spins the homogenate at progressively higher speeds so that the largest organelles come out first.

Autoradiography goes the other way. It leaves the cell intact, labels a molecule radioactively, and lets the radiation expose a photographic emulsion so you can see where that molecule went.

X-ray crystallography answers a different question again: not where a molecule is, but how its atoms are arranged.

1. Cell fractionation

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Cell fractionation was discovered by Albert Claude.

It is the process of separating cellular components while preserving the individual functions of each component, in order to study organelles and different parts of the cell.

The point of the phrase preserving individual functions is that a fraction which no longer works is a fraction you cannot study. Everything about the method is arranged so that the organelles survive being removed from the cell.

Homogenisation

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The first stage breaks the cells open:

  • The tissue sample is placed in a homogeniser, a glass tube.
  • It is covered with an isotonic buffer solution.
  • The plunger is pushed up and down to break open the cells.
  • What is left is the homogenate.

The buffer does two jobs, and both are worth stating separately:

  1. It keeps the pH constant.
  2. It prevents water moving into the organelles by osmosis and bursting them. This is what isotonic is there for: a solution of the wrong strength would destroy the organelles in the act of releasing them.

The homogenate is no longer a cell. It contains all the organelles that we find in cells, free in suspension.

Purification: fractionation by centrifugation

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The second stage separates the homogenate, and it is done by centrifugation. The organelles are separated based on size.

  • Larger organelles, such as nuclei, experience greater force and move towards the bottom of the tube.
  • The supernatant is transferred into a new tube and centrifuged at a higher speed.
  • The process is repeated with a much higher speed again.

So the order in which things come out of suspension runs from the biggest at the lowest speed to the smallest at the fastest:

SpinComes down
Low speedNucleus
FasterMitochondrion
Faster stillLysosomes
Fast speed spinRibosomes

The rule to hold on to is that bigger organelles need a slower spin, because they already experience greater force.

2. Autoradiography

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Autoradiography is a technique to visualise molecules, or fragments of molecules, that have been radioactively labelled.

  • Amino acids or nucleotides are labelled by a radioactive substance built into their molecular structure.
  • The radioactively labelled metabolites, that is the nucleotides and amino acids, emit weak radiation to those regions where the molecules are located, and bind to the selected part of the cell.
  • The cells or tissue sections are then coated, in a darkroom, with photographic emulsion containing silver bromide crystals, which act as detectors for the radiation.
  • Finally the slides are developed photographically.

The darkroom is not a detail. The emulsion is sensitive to light as well as to radiation, so any light reaching it before development would blacken it everywhere and destroy the result.

3. X-ray crystallography

note

X-ray crystallography is a method to determine the arrangement of atoms and the molecular structure of crystals. It is the most favoured technique for the structure determination of proteins.

It is used to see the crystal structure of:

  • proteins;
  • cholesterol and vitamin B12;
  • haemoglobin and myoglobin.

The process:

  • The target substance is mounted in the crystallography machine and illuminated with a beam of X-rays.
  • The electrons in the structure scatter the X-rays into a diffraction pattern.
  • The result is a 3D model in which the atoms and molecules are visible.

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