80
3.
FIXATION
of thin-walled capillary, a little bit longer than the specimen is thick.
This is filled with a colored salt solution which is frozen along with the
specimen. The retreat of the interface can be followed during substitution, and serves to indicate its rate. If riboflavine phosphate is the dye
used, dissolved in a glycerinated salt solution resembling the specimen,
one can tell whether or not crystalline ice exists, for in the vitreous state
this substance shows a brilliant yellow ultraviolet fluorescence, while
crystals have an orange phosphorescence.
It is obvious that the general method of freeze-substitution offers a
flexible technique adaptable to a wide variety of research problems. Much
can be expected of it in years to come. It is not a routine method as yet,
however, and the investigator who would begin working in this field
will have to show resourcefulness in working out technical details.
LITERATURE CITED
Afzelius, B. A. (1959). /. Biophys.
Biochem.
Cytol. 5, 269.
Afzelius, B. A. (1962). In "The Interpretation of Ultrastructure" (R. J. C. Harris, ed.),
p. 1. Academic Press, New York.
Bartl, P. (1962). In "Electron Microscopy" (Proc. 5th Intern. Congr. Electron Microscopy),
Vol. 2, p. P-4. Academic Press, New York.
Bell, L. G. E. (1956). In "Physical Techniques in Biological Research" (G. Oster and
A. W. Pollister, eds.), Vol. Ill, Chapter 1. Academic Press, New York.
Bennett, H. S., and Luft, J. H. (1959). /. Biophys.
Biochem.
Cytol. 6, 113.
Bullivant, S. (1960). /. Biophys.
Biochem.
Cytol. 8, 639.
Caulfield, J. B. (1957). /. Biophys.
Biochem.
Cytol. 3, 827.
Clark, W. M. (1928). "The Determination of Hydrogen Ions," 3rd ed. Williams &
Wilkins Co., Baltimore, Maryland.
Claude, A. (1962). "Electron Microscopy" (Proc. 5th Intern. Congr. Electron Microscopy),
Vol. 2, p. L-14. Academic Press, New York.
Claude, A., and Fullam, E. F. (1946). J. Exptl.
Med. 83, 491.
Dalton, A. J. (1955). Anat. Record
121, 281.
Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. (1950). /. Natl. Cancer Inst. 11,
439.
Ehrlich, H. G. (1958). Exptl.
Cell Research
15, 463.
Elfvin, L. G. (1963). /. Ultrastructure
Research
8, 283.
Feder, N., and Sidman, R. L. (1958). J. Biophys.
Biochem.
Cytol. 4, 593.
Fernandez-Moran, H. (1960). Ann. N. Y. Acad. Sci. 85, 689.
Glick, D., and Malmstrom, B. G. (1952). Exptl.
Cell Research
3, 125.
Grunbaum, B. W., and Wellings, S. R. (1960). /. Ultrastructure
Research
4, 117.
Haarveld, A. van, and Cowell, J. (1964). Anat. Record
(submitted for publication).
Hanzon, V., and Hermodsson, L. H. (1960). /. Ultrastructure
Research
4, 332.
Harris, R. J. C , ed. (1954)." Biological Applications of Freezing and Drying." Academic
Press, New York.
3.
FIXATION
of thin-walled capillary, a little bit longer than the specimen is thick.
This is filled with a colored salt solution which is frozen along with the
specimen. The retreat of the interface can be followed during substitution, and serves to indicate its rate. If riboflavine phosphate is the dye
used, dissolved in a glycerinated salt solution resembling the specimen,
one can tell whether or not crystalline ice exists, for in the vitreous state
this substance shows a brilliant yellow ultraviolet fluorescence, while
crystals have an orange phosphorescence.
It is obvious that the general method of freeze-substitution offers a
flexible technique adaptable to a wide variety of research problems. Much
can be expected of it in years to come. It is not a routine method as yet,
however, and the investigator who would begin working in this field
will have to show resourcefulness in working out technical details.
LITERATURE CITED
Afzelius, B. A. (1959). /. Biophys.
Biochem.
Cytol. 5, 269.
Afzelius, B. A. (1962). In "The Interpretation of Ultrastructure" (R. J. C. Harris, ed.),
p. 1. Academic Press, New York.
Bartl, P. (1962). In "Electron Microscopy" (Proc. 5th Intern. Congr. Electron Microscopy),
Vol. 2, p. P-4. Academic Press, New York.
Bell, L. G. E. (1956). In "Physical Techniques in Biological Research" (G. Oster and
A. W. Pollister, eds.), Vol. Ill, Chapter 1. Academic Press, New York.
Bennett, H. S., and Luft, J. H. (1959). /. Biophys.
Biochem.
Cytol. 6, 113.
Bullivant, S. (1960). /. Biophys.
Biochem.
Cytol. 8, 639.
Caulfield, J. B. (1957). /. Biophys.
Biochem.
Cytol. 3, 827.
Clark, W. M. (1928). "The Determination of Hydrogen Ions," 3rd ed. Williams &
Wilkins Co., Baltimore, Maryland.
Claude, A. (1962). "Electron Microscopy" (Proc. 5th Intern. Congr. Electron Microscopy),
Vol. 2, p. L-14. Academic Press, New York.
Claude, A., and Fullam, E. F. (1946). J. Exptl.
Med. 83, 491.
Dalton, A. J. (1955). Anat. Record
121, 281.
Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. (1950). /. Natl. Cancer Inst. 11,
439.
Ehrlich, H. G. (1958). Exptl.
Cell Research
15, 463.
Elfvin, L. G. (1963). /. Ultrastructure
Research
8, 283.
Feder, N., and Sidman, R. L. (1958). J. Biophys.
Biochem.
Cytol. 4, 593.
Fernandez-Moran, H. (1960). Ann. N. Y. Acad. Sci. 85, 689.
Glick, D., and Malmstrom, B. G. (1952). Exptl.
Cell Research
3, 125.
Grunbaum, B. W., and Wellings, S. R. (1960). /. Ultrastructure
Research
4, 117.
Haarveld, A. van, and Cowell, J. (1964). Anat. Record
(submitted for publication).
Hanzon, V., and Hermodsson, L. H. (1960). /. Ultrastructure
Research
4, 332.
Harris, R. J. C , ed. (1954)." Biological Applications of Freezing and Drying." Academic
Press, New York.