Radio labelled Tracers (Radio labelled compound)

When one or more atom of chemical compound replaced by radioisotopes used- for the study of the biosynthetic pathway, is known as Radiotracers.

Radiotracer Technique:

The technique which utilises radioactive labelled compound to find out or to trace various precursors and intermediates involved at different stages of biosynthetic pathway at given rate and time.

In this technique, different isotope, mainly the radioactive isotopes which are incorporated into the presumed precursor of plant metabolites & used as marker in the biogenic studies.


 

Steps in Tracer Technique


1.                  Selection of Radioisotopes

2.                  Preparation of Radioisotopes.

3.                  Introduction/Insertion of Radiolabelled compound in biological system (Plant part).

4.                  Seperation and determination of labelled compound in various biochemical reaction


Detection & assay of Radioactive labelled compound

Detectors system used (Analysis of Isotopic content)

(a)     Geiger -Muller Counter- Detection and measurement of all types of radiation

(b)    Liquid Scintillation Counter – Scintillators are used (For Beta emitting radioisotopes)

(c)     Autoradiography - to trace the location of radioactive isotope in biological system

 

(a) Geiger Muller Counter

Geiger counters are used to detect radioactive emissions, most commonly beta particles and gamma rays.

The counter consists of a tube filled with an inert gas that becomes conductive of electricity when it is impacted by a high-energy particle.

When a Geiger counter is exposed to ionizing radiation, The particles penetrate the tube and collide with the gas,  releasing more electrons.

Positive ions exit the tube and the negatively charged electrons become attracted to a high-voltage middle wire.

When the number of electrons that build up around the wire reaches a threshold, it creates an electric current.


This causes the temporary closing of a switch and generates an electric pulse that is registered on a meter, either acoustically as a click that increases in intensity as the ionizing

radiation increases, or visually as  the motion of a needle pointer.

 

(b) Liquid Scintillation Counter


Basically, the liquid scintillation process is the conversion of the energy of a radioactive decay event into photons of light in a liquid.

Photomultipliers (PM-tubes) detect the emission of light and convert the light pulse into an electrical signal.

The intensity of the light pulse (number of photons emitted) is proportional to the energy of the radioactive decay event.

Further, the size (height) of the electrical pulse is proportional to the intensity of the light and, accordingly, also proportional to the energy of  the decay event.



 The electrical pulse can be handled in an electronic system, which measures its height and stores the events in an intensity-energy array, also-called multichannel analyzer (MCA) system.

Thus, an energy spectrum can be recorded of the decaying radionuclide. 

In this way liquid scintillation counting (LSC) is a detection technique for radioactivity. Normally, the radioactive substance is intimately mixed with the detector which is the liquid scintillator cocktail.

The radioactive substance should  then, preferably, be in liquid form.

The emission of light in the liquid scintillator is an isotropic process.

By applying two PM tube (Photo multiplier-tubes) instead of only one the noise in the detection process (background counts not due to the decay process) may then be reduced.

Only those events that are recorded in both PM-tubes simultaneously (in coincidence within the  required time window, often 10-30 ns width) are recorded as “true” counts. 

 

(C) Auto-radiography


Autoradiography is the bio-analytical technique used to visualize the distribution of radioactive labeled substance with radioisotope in a biological sample.

It is a method by which a radioactive material can be localized within a particular tissue, cell, cell organelles or even biomolecules.



It is very sensitive technique and is being used in a wide variety of biological experiments.

Autoradiography, although used to locate the radioactive substances, it can also be used for quantitative estimation by using densitometer.

  

Principle

Autoradiography is based upon the ability of radioactive substance to expose the photographic film by ionizing it.

In this technique a radioactive substance is put in direct contact with a thick layer of a photographic emulsion (thickness of 5-50 μm) having gelatin substances and silver halide crystals.

This emulsion differs from the standard photographic film in terms of having higher ratio of silver halide to gelatin and small size of grain. It is then left in dark for several days for proper exposure.

The silver halide crystals are exposed to the radiation which chemically converts silver halide into metallic silver (reduced) giving a dark color band.

The resulting radiography is viewed by electron microscope, preflashed screen, intensifying screen, electrophoresis, digital scanners etc.

 

Methodology

The radioactive sample is covered with the photographic emulsion by several described method.

The radioactive part of the sample activates the silver halide crystals near by.

This results in reduction of Ag' ions to Ag atom leaving dark color bands.

The slide is then washed away by fixers to get insoluble  Ag atom only.

The autoradiogram can further be viewed and observed under the microscope.


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