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Chemical coating and chemical dehydration of biological specimens for scanning electron microscopy
Micron, 1981, Vol.: 12, pp.181-182. ~ergamon Press Ltd. Printed in Great Britain.
0047-7206/81/020181-O2502.00/0
CHEMICAL COATING AND CHEMICAL DEHYDRATION OF BIOLOGICAL SPECIMENS FOR SCANNING ELECTRON MICROSCOPY
D.J. Hall and W.D.E. Thomas Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BSI8 9AF.
In the OTO process proposed by Postek and Tucker (1977), charging of biological specimens during scanning is minimised by adopting a ligand osmium binding technique utilising thiocarbohydrazide (TCH), as an alternative to evaporation or sputter coating. The osmium/ TCH procedure, though tedious, does increase tissue conductivity and so reduces charging but can result in areas of the surface being covered with particulate matter (Fig. la). By modifying the process slightly (using 0.5% TCH for I h instead of I% (saturated) TCH for 30 min) specimens with surfaces free of contamination are obtained so giving acceptable images (Fig. Ib). The chemical dehydration of biological tissue using 2,2-dimethoxypropane (DMP) proposed by Muller and Jacks (1975) has been investigated. Although the reaction between DMP and water is rapid, we have found preservation to be very dependent on the time of in~nersion in DMP. With optimum inmlersion time - and this varies with type and size of specimen - preservation of the specimen after subsequent critical point drying (Hall et al., 1978) was as good as if the specimen had been dehydrated conventionally by solvent substitution (Figs. 2a and b).
REFERENCES Hall, D.J., Skerrett, E.J. and Thomas, W.D.E., 1978. Critical point drying for scanning electron microscopy: a semi-automatic method of preparing biological specimens. J. Microsc. 113: 277-290. Muller, L.L. and Jacks, T.J., 1975. Rapid chemical dehydration of samples for electron microscopic examination. J. Histochem. Cytochem. 23: 107-110. Postek, M.T. and Tucker, Shirley C., 1977. Thiocarbohydrazide binding for botanical specimens for scanning electron microscopy: a modification. J. Microsc. 110: 71-74.
181
182
D..I.
Fig. 1
Hall
and W.D.E.
Thomas
Pelargonium zonale leaf (abaxial surface) after
(a) impregnation with OsO 4 and ]% (satd. soln.) TCH for 30 min (OTO process) followed by solvent dehydration and CPD, Charging minimal but specimen surface covered with particulate matterl (b) procedure as in Ca) but using TCH at 0.5% for I h. Charging minimal and specimen free of particulate matter.
Fig. 2
Pelargonium zonale leaf (abaxial surface) after
(a) immersion for 15 min in DMP followed by CPD and sputter coating. Good preservation; (b)
immersion for 16 min in DMP and then as in (a).
Extensive damage.
0047-7206/81/020181-O2502.00/0
CHEMICAL COATING AND CHEMICAL DEHYDRATION OF BIOLOGICAL SPECIMENS FOR SCANNING ELECTRON MICROSCOPY
D.J. Hall and W.D.E. Thomas Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BSI8 9AF.
In the OTO process proposed by Postek and Tucker (1977), charging of biological specimens during scanning is minimised by adopting a ligand osmium binding technique utilising thiocarbohydrazide (TCH), as an alternative to evaporation or sputter coating. The osmium/ TCH procedure, though tedious, does increase tissue conductivity and so reduces charging but can result in areas of the surface being covered with particulate matter (Fig. la). By modifying the process slightly (using 0.5% TCH for I h instead of I% (saturated) TCH for 30 min) specimens with surfaces free of contamination are obtained so giving acceptable images (Fig. Ib). The chemical dehydration of biological tissue using 2,2-dimethoxypropane (DMP) proposed by Muller and Jacks (1975) has been investigated. Although the reaction between DMP and water is rapid, we have found preservation to be very dependent on the time of in~nersion in DMP. With optimum inmlersion time - and this varies with type and size of specimen - preservation of the specimen after subsequent critical point drying (Hall et al., 1978) was as good as if the specimen had been dehydrated conventionally by solvent substitution (Figs. 2a and b).
REFERENCES Hall, D.J., Skerrett, E.J. and Thomas, W.D.E., 1978. Critical point drying for scanning electron microscopy: a semi-automatic method of preparing biological specimens. J. Microsc. 113: 277-290. Muller, L.L. and Jacks, T.J., 1975. Rapid chemical dehydration of samples for electron microscopic examination. J. Histochem. Cytochem. 23: 107-110. Postek, M.T. and Tucker, Shirley C., 1977. Thiocarbohydrazide binding for botanical specimens for scanning electron microscopy: a modification. J. Microsc. 110: 71-74.
181
182
D..I.
Fig. 1
Hall
and W.D.E.
Thomas
Pelargonium zonale leaf (abaxial surface) after
(a) impregnation with OsO 4 and ]% (satd. soln.) TCH for 30 min (OTO process) followed by solvent dehydration and CPD, Charging minimal but specimen surface covered with particulate matterl (b) procedure as in Ca) but using TCH at 0.5% for I h. Charging minimal and specimen free of particulate matter.
Fig. 2
Pelargonium zonale leaf (abaxial surface) after
(a) immersion for 15 min in DMP followed by CPD and sputter coating. Good preservation; (b)
immersion for 16 min in DMP and then as in (a).
Extensive damage.