MICRO AUTORADIOGRAPHY

S.MURALI20166020

05

Autoradiography

Autoradiography is the bio-analytical technique used to visualize the distribution of radioactive

labelled 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 a 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.

HISTORY The first autoradiography was obtained accidently around 1867 when a blackening was produced on

emulsions of silver chloride and iodide by uranium salts observed by Niepce de St. Victor.

In 1924 first biological experiment involving autoradiography traced the distribution of polonium in

biological specimens.

The development of autoradiography as a biological technique really started to happen after World war II

with the development of photographic emulsions and then stripping made of silver halide.

Radioactivity is now no longer the property of a few rare elements of minor biological interest (such as

radium, thorium or uranium) as now any biological compound can be labelled with radioactive isotopes

opening up many possibilities in the study of living systems.

MICRO AUTORADIOGRAPHY

Micro autoradiography, has been developed for studying subcellular

structures, even those as small as individual strands of deoxyribonucleic

acid (DNA).

Much interesting information has been learned about the mechanisms of cell

division and other processes in cell biology.

The cells being studied are given a nutrient solution containing molecules that

have been labeled, usually with radioactive tritium, carbon, or phosphorus.

PRINCIPLES OF AUTO RADIOGRAPHY

• Resolution and radioisotope characteristics

• Film emulsion and sensitivity

• Determination of exposure time

• Tissue preparation and artifacts

PRINCIPLE

Incubate tissue withradioactive ligand

Expose to filmor emulsion

Isotope will emitradiation (usually beta)

Radiation will hit silver grains in emulsion and expose them

BASIC COMPONENTS

• Specimen• Tracer• Detector

MICRO AUTORADIOGRAPHY PROCEDURE 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 mm) 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 colour band. The resulting radiography is viewed by electron microscope, pre flashed screen, intensifying screen,

electrophoresis, digital scanners etc.

RECEPTOR MICRO-AUTORADIOGRAPHYSequence of steps APPLICATION of radiolabelled compound with high specific activity (including competition)

EXCISION of multiple tissues and positioning on holders

FREEZE-MOUNTING on tissue holders

LIQUID NITROGEN STORAGE of mounted tissues

CRYO-SECTIONING of 4 lm (or less) sections

THAW-MOUNTING on emulsion-coated slides

PHOTOGRAPHIC EXPOSURE in desiccator boxes

PHOTOGRAPHIC PROCESSING

STAINING with MBBF or MGP

AIR-DRYING and COVER-SLIPPING

EVALUATIONqualitative and quantitative

Applications

To find and investigate the various properties of DNA

To find the location and amount of particular substance within a cell

including cell organelle, metabolites etc.

Tissue localization of radioactive substance.

To find the site and performance of targeted drug.

To locate the metabolic activity site in the cell.

Radioactive precursors of DNA and RNA, [3H]-thymidine and [3H]-

uridine respectively, may be to determine the timing of several phases

of the cell cycle. introduced to living cells

RNA or DNA viral sequences can also be located in this fashion. These

probes are usually labelled with 32P, 33P, or 35S.

Practical Applications

In agricultural research, the effectiveness of herbicides, insecticides, and

fertilizers is studied to determine which ones can increase productivity without

causing serious environmental problems. Radioactive phosphorus can be used in

this regard to study plant metabolism.

The uptake of iron or zinc from the soil and their circulation in a plant can be

studied to ascertain the effect of soil acidity and chemical form. Sometimes the

presence or absence of other elements can inhibit translocation of an essential

nutrient.

New plant growth regulators may move from one plant through the soil to a

nearby untreated plant. Autoradiography is an important analytical technique for

observing the route of micronutrients and discovering what factors can change

their mobility in a plant.

The sequence of bases in DNA molecules can be decoded by using

electrophoresis combined with autoradiography, and the study of DNA sequences

is crucial to research in many diverse areas of biology. Although alternatives to

using autoradiography in DNA sequencing are now common, autoradiography is

still a standard technique used in many other aspects of molecular biology.

Conclusion MARG is a High Resolution Histological Tool to investigate spatial localization of radiolabelled

drugs at a tissue, and cellular level.

Ex vivo and exsanguination occurs

Numerous elaborations on the techniques

Cryo-preservation required for soluble compounds. Liquid tissue fixation (formalin) often solubilizes and relocates diffusible test articles. Exception for receptor-bound TA.

Not Quantitative – No standards used, prone to artefacts, lack of control on detection media and section thickness.

References :

Adamczyk, J., M. Hesselhoe, N. Iversen, M. Horn, A. Lehner, P.H. Nielsen, M. Scholter, P. Roslev, and

M. Wagner. 2003. The isotope array, a new tool that employs substrate mediated labelling of rRNA for

determination of microbial community structure and function. Appl. Environ. Microbiol. 69:6875–

6887.

Thiel, R., and M. Blaut. 2005. An improved method for the automated enumeration of fluorescently

labelled bacteria in human faeces. J. Microbiol. Methods 61:369–379.

Stumpf W E, Sar M, Joshi S G. Estrogen target cells in the skin. Experientia 1974: 30: 196–198.

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