LOW TEMPERATURE ELECTRON MICROSCOPY UNIT

Low temperature methods in electron microscopy have been widespreading through laboratories since the middle of eighties. The general aim of this research is to find methodological approaches that can permit a better preservation of the ultrastructure of biological specimens. Due to the very low pressure existing into the electron microscope after reaching vacuum, the water naturally present in the cell evaporates extremely quickly with disastrous effect on the ultrastructure of the cell.

Processing the cell using physical methods (e.g. low temperature) fixes rapidly the water together with other organic molecules present in the cell, avoiding the artefacts that are commonly produced with conventional chemical fixation.

The low temperature methods employed in our Department for electron microscopy consist of rapid freezing by dipping into liquid propane followed by a substitution step in acetone at –80°C. Our interest focuses on plant material with special attention to the male apparatus of Angiosperms (e.g. pollen). The apparatus used is a hand-made one, constructed on the model of several other commercial devices.

This approach resulted particularly useful for several species (Gingko biloba, Cupressus spp., Corylus avellana, Oryza sativa,), where the conventional methodologies revealed to be difficult to apply. Furthermore this technique should be considered almost indispensable when processing the material for immunogold labelling, due to the reliability of the procedure in preserving biological structures.

For more information see also:

C.H.Theunis, M.Cresti and C.Milanesi. Studies of the mature pollen of Spinacia Oleracea after freeze-substitution and observed with confocal laser scanning fluorescence microscopy, Botan.Acta 104: 324-329 1991

Gamal El-Ghazaly, S.Nakamura, Y.Takahashi, M. Cresti, Bjorn Walles and C. Milanesi. Localisation of the major allergen Bet v 1 in Betula pollen using monoclonal antibody labelling, Grana 35: 369-374 1996

C.Milanesi, A.Moscatelli and M.Cresti. Tips for freeze-substitution and immunogold labelling pollen grain from different species, Cryo 97 6-9 luglio 1997 York (UK)

JOANNE H. E. ROSS, CLAUDIO MILANESI, DENIS J . MURPHY, MAURO CRESTI:
Rapid freeze fixation and substitution improved tissue preservation of microspores and tapetum in Brassica Napus.
Sex Plant Reproduction 12: 237-240 2000
 

For contact: send an e-mail to Claudio Milanesi
 
 
 

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