Abstract

<p>The development of phase contrast and interference microscopy has focused attention on the possibility of measuring the refractive indexes of living cells. Difficulties are encountered in applying immersion methods of refractometry to living cells because the choice of immersion media is very limited. These difficulties have now been overcome by the use of protein solutions. When the refractive index of the immersion medium matches that of the cell cytoplasm the latter becomes virtually invisible and phase contrast can be used as a sensitive null indicator to determine the match position. The refractive index is directly related to the concentration of cell solids and refractometry has been extensively used for measuring such concentrations in a variety of living cells. The concentration of water also follows immediately. Examples of the application of this method to retinal rods and cones, to the study of cell populations such as red blood cells, and to the investigation of the osmotic reactions of living cells are given. Other applications such as contrast variation in living cells, demonstration of chromosomes, and the spectrophotometry of clarified cell suspensions are briefly discussed.</p><p>Interference microscopy can be used for the direct measurement of optical path difference and it is shown that this quantity is directly related to the dry mass per unit area. By combining interference microscopy and immersion refractometry it becomes possible to measure the cell thickness and hence to calculate dry mass and wet mass, volume, and water content of living cells. Examples of these methods are given and some of the limitations indicated.</p>

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  2. H. H. Pfeiffer, Cellule 54, 41 (1951).
  3. F. Vles, Compt. rend. soc. biol. 85, 494 (1921).
  4. E. S. Castle, J. Gen. Physiol. 17, 41 (1934).
  5. S. Exner, Pflügers Arch. ges. Physiol. 40, 379 (1887).
  6. E. Fauré-Frémiet, Protoplasma 6, 521 (1929).
  7. R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).
  8. Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
  9. R. Barer and S. Joseph, Quart. J. Microscop. Sci. 96, 1 (1955).
  10. M. Françon, Le Contraste de Phase (Editions de la Revue d'Optique, Paris, 1950).
  11. R. Barer, J. Roy. Microscop. Soc. 72, 10 (1952).
  12. R. Barer and S. Joseph (to be published).
  13. R. Barer and S. Joseph, Quart. J. Microscop. Sci. 95, 399 (1954).
  14. W. D. Wright and J. H. Nelson, Proc. Phys. Soc. (London) 48, 401 (1936).
  15. B. O'Brien, J. Opt. Soc. Am. 41, 882 (1951).
  16. W. S. Stiles and B. H. Crawford, Proc. Roy. Soc. (London) B112, 428 (1923).
  17. R. L. Sidman and G. B. Wislocki, J. Histochem. Cytochem. 2, 413 (1954).
  18. G. Toraldo di Francia, J. Opt. Soc. Am. 39, 324 (1949).
  19. G. Toraldo di Francia, Proc. Phys. Soc. (London) B62, 461 (1949).
  20. W. E. Le Gros Clark, Documenta Ophthalmol. 3, 57 (1949).
  21. G. Wald, Documenta Ophthalmol. 3, 94 (1949).
  22. W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London 1946).
  23. W. de W. Abney, Researches in Colour Vision (Longmans Green and Company, London, 1913).
  24. F. H. G. Pitt, Proc. Roy. Soc. (London) B131, 101 (1944).
  25. A. Frey-Wyssling, Naturwissenschaften 40, 517 (1953).
  26. R. Barer, Naturwissenschaften 41, 206 (1954).
  27. R. Barer, Science 121, 709 (1955).
  28. R. Barer and R. L. Sidman, J. Physiol. 129, 60P (1955).
  29. J. Dyson, Proc. Roy. Soc. (London) A204, 170 (1950).
  30. F. H. Smith, Brit. Pat. Spec. 6,39,014 (1950); Research (London) 8, 385 (1955).
  31. M. Françon, Le Microscope à Contraste de Phase et le Microscope Interférentiel (Editions du C. N. R. S., Paris, 1954).
  32. R. Barer, in Analytical Cytology, edited by R. C. Mellors (McGraw-Hill Book Company, Inc., New York, 1955).
  33. R. Barer, Nature 169, 366 (1952).
  34. H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).
  35. Davies, Wilkins, Chayen, and La Cour, Quart. J. Microscop. Sci. 95, 271 (1954).
  36. Barter, Danielli, and Davies, Proc. Roy. Soc. (London) B144, 412 (1955).
  37. A. J. Hale, Exptl. Cell Research 10, 132 (1956).
  38. T. Caspersson, Experientia 11, 45 (1955).
  39. Mellors, Stoholski, and Beyer, Cancer 7, 813 (1954).
  40. R. Barer, Nature 172, 1097 (1953).
  41. Dossier, Jacquinot, Maréchal, and Pieuchard, J. phys. radium 12, 565 (1951).
  42. R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

1956 (1)

A. J. Hale, Exptl. Cell Research 10, 132 (1956).

1955 (6)

T. Caspersson, Experientia 11, 45 (1955).

Barter, Danielli, and Davies, Proc. Roy. Soc. (London) B144, 412 (1955).

R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

R. Barer, Science 121, 709 (1955).

R. Barer and R. L. Sidman, J. Physiol. 129, 60P (1955).

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 96, 1 (1955).

1954 (5)

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 95, 399 (1954).

R. L. Sidman and G. B. Wislocki, J. Histochem. Cytochem. 2, 413 (1954).

R. Barer, Naturwissenschaften 41, 206 (1954).

Davies, Wilkins, Chayen, and La Cour, Quart. J. Microscop. Sci. 95, 271 (1954).

Mellors, Stoholski, and Beyer, Cancer 7, 813 (1954).

1953 (3)

R. Barer, Nature 172, 1097 (1953).

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).

A. Frey-Wyssling, Naturwissenschaften 40, 517 (1953).

1952 (4)

R. Barer, Nature 169, 366 (1952).

H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).

R. Barer, J. Roy. Microscop. Soc. 72, 10 (1952).

R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).

1951 (3)

H. H. Pfeiffer, Cellule 54, 41 (1951).

B. O'Brien, J. Opt. Soc. Am. 41, 882 (1951).

Dossier, Jacquinot, Maréchal, and Pieuchard, J. phys. radium 12, 565 (1951).

1950 (2)

J. Dyson, Proc. Roy. Soc. (London) A204, 170 (1950).

F. H. Smith, Brit. Pat. Spec. 6,39,014 (1950); Research (London) 8, 385 (1955).

1949 (4)

G. Toraldo di Francia, J. Opt. Soc. Am. 39, 324 (1949).

G. Toraldo di Francia, Proc. Phys. Soc. (London) B62, 461 (1949).

W. E. Le Gros Clark, Documenta Ophthalmol. 3, 57 (1949).

G. Wald, Documenta Ophthalmol. 3, 94 (1949).

1944 (1)

F. H. G. Pitt, Proc. Roy. Soc. (London) B131, 101 (1944).

1936 (1)

W. D. Wright and J. H. Nelson, Proc. Phys. Soc. (London) 48, 401 (1936).

1934 (1)

E. S. Castle, J. Gen. Physiol. 17, 41 (1934).

1929 (1)

E. Fauré-Frémiet, Protoplasma 6, 521 (1929).

1923 (1)

W. S. Stiles and B. H. Crawford, Proc. Roy. Soc. (London) B112, 428 (1923).

1921 (1)

F. Vles, Compt. rend. soc. biol. 85, 494 (1921).

1887 (1)

S. Exner, Pflügers Arch. ges. Physiol. 40, 379 (1887).

Barer, R.

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 96, 1 (1955).

R. Barer, Science 121, 709 (1955).

R. Barer and R. L. Sidman, J. Physiol. 129, 60P (1955).

R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

R. Barer, Naturwissenschaften 41, 206 (1954).

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 95, 399 (1954).

R. Barer, Nature 172, 1097 (1953).

R. Barer, J. Roy. Microscop. Soc. 72, 10 (1952).

R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).

R. Barer, Nature 169, 366 (1952).

R. Barer, in Physical Techniques in Biological Research, edited by Oster and Pollister (Academic Press, Inc., New York, 1956), Vol. III.

R. Barer and S. Joseph (to be published).

R. Barer, in Analytical Cytology, edited by R. C. Mellors (McGraw-Hill Book Company, Inc., New York, 1955).

Caspersson, T.

T. Caspersson, Experientia 11, 45 (1955).

Castle, E. S.

E. S. Castle, J. Gen. Physiol. 17, 41 (1934).

Clark, W. E. Le Gros

W. E. Le Gros Clark, Documenta Ophthalmol. 3, 57 (1949).

Cour, La

Davies, Wilkins, Chayen, and La Cour, Quart. J. Microscop. Sci. 95, 271 (1954).

Crawford, B. H.

W. S. Stiles and B. H. Crawford, Proc. Roy. Soc. (London) B112, 428 (1923).

Davies, H. G.

H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).

de W. Abney, W.

W. de W. Abney, Researches in Colour Vision (Longmans Green and Company, London, 1913).

di Francia, G. Toraldo

G. Toraldo di Francia, J. Opt. Soc. Am. 39, 324 (1949).

G. Toraldo di Francia, Proc. Phys. Soc. (London) B62, 461 (1949).

Dick, D. A. T.

R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

Dyson, J.

J. Dyson, Proc. Roy. Soc. (London) A204, 170 (1950).

Exner, S.

S. Exner, Pflügers Arch. ges. Physiol. 40, 379 (1887).

Fauré-Frémiet, E.

E. Fauré-Frémiet, Protoplasma 6, 521 (1929).

Françon, M.

M. Françon, Le Microscope à Contraste de Phase et le Microscope Interférentiel (Editions du C. N. R. S., Paris, 1954).

M. Françon, Le Contraste de Phase (Editions de la Revue d'Optique, Paris, 1950).

Frey-Wyssling, A.

A. Frey-Wyssling, Naturwissenschaften 40, 517 (1953).

Hale, A. J.

A. J. Hale, Exptl. Cell Research 10, 132 (1956).

Joseph, S.

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 96, 1 (1955).

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 95, 399 (1954).

R. Barer and S. Joseph (to be published).

Nelson, J. H.

W. D. Wright and J. H. Nelson, Proc. Phys. Soc. (London) 48, 401 (1936).

O’Brien, B.

B. O'Brien, J. Opt. Soc. Am. 41, 882 (1951).

Pfeiffer, H. H.

H. H. Pfeiffer, Cellule 54, 41 (1951).

Pitt, F. H. G.

F. H. G. Pitt, Proc. Roy. Soc. (London) B131, 101 (1944).

Ross, K. F. A.

R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).

Sidman, R. L.

R. Barer and R. L. Sidman, J. Physiol. 129, 60P (1955).

R. L. Sidman and G. B. Wislocki, J. Histochem. Cytochem. 2, 413 (1954).

Smith, F. H.

F. H. Smith, Brit. Pat. Spec. 6,39,014 (1950); Research (London) 8, 385 (1955).

Stiles, W. S.

W. S. Stiles and B. H. Crawford, Proc. Roy. Soc. (London) B112, 428 (1923).

Vles, F.

F. Vles, Compt. rend. soc. biol. 85, 494 (1921).

Wald, G.

G. Wald, Documenta Ophthalmol. 3, 94 (1949).

Wilkins, M. H. F.

H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).

Wislocki, G. B.

R. L. Sidman and G. B. Wislocki, J. Histochem. Cytochem. 2, 413 (1954).

Wright, W. D.

W. D. Wright and J. H. Nelson, Proc. Phys. Soc. (London) 48, 401 (1936).

W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London 1946).

Other (42)

R. Barer, in Physical Techniques in Biological Research, edited by Oster and Pollister (Academic Press, Inc., New York, 1956), Vol. III.

H. H. Pfeiffer, Cellule 54, 41 (1951).

F. Vles, Compt. rend. soc. biol. 85, 494 (1921).

E. S. Castle, J. Gen. Physiol. 17, 41 (1934).

S. Exner, Pflügers Arch. ges. Physiol. 40, 379 (1887).

E. Fauré-Frémiet, Protoplasma 6, 521 (1929).

R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 96, 1 (1955).

M. Françon, Le Contraste de Phase (Editions de la Revue d'Optique, Paris, 1950).

R. Barer, J. Roy. Microscop. Soc. 72, 10 (1952).

R. Barer and S. Joseph (to be published).

R. Barer and S. Joseph, Quart. J. Microscop. Sci. 95, 399 (1954).

W. D. Wright and J. H. Nelson, Proc. Phys. Soc. (London) 48, 401 (1936).

B. O'Brien, J. Opt. Soc. Am. 41, 882 (1951).

W. S. Stiles and B. H. Crawford, Proc. Roy. Soc. (London) B112, 428 (1923).

R. L. Sidman and G. B. Wislocki, J. Histochem. Cytochem. 2, 413 (1954).

G. Toraldo di Francia, J. Opt. Soc. Am. 39, 324 (1949).

G. Toraldo di Francia, Proc. Phys. Soc. (London) B62, 461 (1949).

W. E. Le Gros Clark, Documenta Ophthalmol. 3, 57 (1949).

G. Wald, Documenta Ophthalmol. 3, 94 (1949).

W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London 1946).

W. de W. Abney, Researches in Colour Vision (Longmans Green and Company, London, 1913).

F. H. G. Pitt, Proc. Roy. Soc. (London) B131, 101 (1944).

A. Frey-Wyssling, Naturwissenschaften 40, 517 (1953).

R. Barer, Naturwissenschaften 41, 206 (1954).

R. Barer, Science 121, 709 (1955).

R. Barer and R. L. Sidman, J. Physiol. 129, 60P (1955).

J. Dyson, Proc. Roy. Soc. (London) A204, 170 (1950).

F. H. Smith, Brit. Pat. Spec. 6,39,014 (1950); Research (London) 8, 385 (1955).

M. Françon, Le Microscope à Contraste de Phase et le Microscope Interférentiel (Editions du C. N. R. S., Paris, 1954).

R. Barer, in Analytical Cytology, edited by R. C. Mellors (McGraw-Hill Book Company, Inc., New York, 1955).

R. Barer, Nature 169, 366 (1952).

H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).

Davies, Wilkins, Chayen, and La Cour, Quart. J. Microscop. Sci. 95, 271 (1954).

Barter, Danielli, and Davies, Proc. Roy. Soc. (London) B144, 412 (1955).

A. J. Hale, Exptl. Cell Research 10, 132 (1956).

T. Caspersson, Experientia 11, 45 (1955).

Mellors, Stoholski, and Beyer, Cancer 7, 813 (1954).

R. Barer, Nature 172, 1097 (1953).

Dossier, Jacquinot, Maréchal, and Pieuchard, J. phys. radium 12, 565 (1951).

R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

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