Abstract

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.

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.

© 1957 Optical Society of America

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References

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  1. R. Barer, in Physical Techniques in Biological Research, edited by Oster and Pollister (Academic Press, Inc., New York, 1956), Vol. III.
  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).
    [CrossRef]
  5. S. Exner, Pflügers Arch. ges. Physiol. 40, 379 (1887).
  6. E. Fauré-Frémiet, Protoplasma 6, 521 (1929).
    [CrossRef]
  7. R. Barer and K. F. A. Ross, J. Physiol. 118, 38P (1952).
  8. Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
    [CrossRef] [PubMed]
  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).
    [CrossRef]
  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).
    [CrossRef]
  15. B. O’Brien, J. Opt. Soc. Am. 41, 882 (1951).
    [CrossRef]
  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).
    [CrossRef] [PubMed]
  18. G. Toraldo di Francia, J. Opt. Soc. Am. 39, 324 (1949).
    [CrossRef]
  19. G. Toraldo di Francia, Proc. Phys. Soc. (London) B62, 461 (1949).
  20. W. E. Le Gros Clark, Documenta Ophthalmol. 3, 57 (1949).
    [CrossRef]
  21. G. Wald, Documenta Ophthalmol. 3, 94 (1949).
    [CrossRef]
  22. W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London1946).
  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).
    [CrossRef]
  26. R. Barer, Naturwissenschaften 41, 206 (1954).
    [CrossRef]
  27. R. Barer, Science 121, 709 (1955).
    [CrossRef] [PubMed]
  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).
    [CrossRef] [PubMed]
  34. H. G. Davies and M. H. F. Wilkins, Nature 169, 541 (1952).
    [CrossRef]
  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).
    [CrossRef]
  38. T. Caspersson, Experientia 11, 45 (1955).
    [CrossRef] [PubMed]
  39. Mellors, Stoholski, and Beyer, Cancer 7, 813 (1954).
  40. R. Barer, Nature 172, 1097 (1953).
    [CrossRef] [PubMed]
  41. Dossier, Jacquinot, Maréchal, and Pieuchard, J. phys. radium 12, 565 (1951).
    [CrossRef]
  42. R. Barer and D. A. T. Dick, J. Physiol. 128, 25P (1955).

1956 (1)

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

1955 (6)

T. Caspersson, Experientia 11, 45 (1955).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

R. Barer, Naturwissenschaften 41, 206 (1954).
[CrossRef]

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).
[CrossRef] [PubMed]

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
[CrossRef] [PubMed]

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

1952 (4)

R. Barer, Nature 169, 366 (1952).
[CrossRef] [PubMed]

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

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

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).
[CrossRef]

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

1950 (1)

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

1949 (4)

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

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

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

G. Wald, Documenta Ophthalmol. 3, 94 (1949).
[CrossRef]

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).
[CrossRef]

1934 (1)

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

1929 (1)

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

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).

Abney, W. de W.

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

Barer,

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
[CrossRef] [PubMed]

Barer, R.

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

R. Barer, Science 121, 709 (1955).
[CrossRef] [PubMed]

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).
[CrossRef]

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

R. Barer, Nature 172, 1097 (1953).
[CrossRef] [PubMed]

R. Barer, Nature 169, 366 (1952).
[CrossRef] [PubMed]

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

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

Barter,

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

Beyer,

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

Caspersson, T.

T. Caspersson, Experientia 11, 45 (1955).
[CrossRef] [PubMed]

Castle, E. S.

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

Chayen,

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).

Danielli,

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

Davies,

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

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

Davies, H. G.

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

Dick, D. A. T.

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

Dossier,

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

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).
[CrossRef]

Françon, M.

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

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

Frey-Wyssling, A.

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

Hale, A. J.

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

Jacquinot,

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

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).

La Cour,

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

Le Gros Clark, W. E.

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

Maréchal,

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

Mellors,

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

Nelson, J. H.

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

O’Brien, B.

Pfeiffer, H. H.

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

Pieuchard,

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

Pitt, F. H. G.

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

Ross,

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
[CrossRef] [PubMed]

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).
[CrossRef] [PubMed]

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).

Stoholski,

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

Tkaczyk,

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
[CrossRef] [PubMed]

Toraldo di Francia, G.

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

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

Vles, F.

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

Wald, G.

G. Wald, Documenta Ophthalmol. 3, 94 (1949).
[CrossRef]

Wilkins,

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

Wilkins, M. H. F.

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

Wislocki, G. B.

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

Wright, W. D.

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

W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London1946).

Cancer (1)

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

Cellule (1)

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

Compt. rend. soc. biol. (1)

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

Documenta Ophthalmol. (2)

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

G. Wald, Documenta Ophthalmol. 3, 94 (1949).
[CrossRef]

Experientia (1)

T. Caspersson, Experientia 11, 45 (1955).
[CrossRef] [PubMed]

Exptl. Cell Research (1)

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

J. Gen. Physiol. (1)

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

J. Histochem. Cytochem. (1)

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

J. Opt. Soc. Am. (2)

J. phys. radium (1)

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

J. Physiol. (3)

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

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

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

J. Roy. Microscop. Soc. (1)

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

Nature (4)

Barer, Ross, and Tkaczyk, Nature 171, 720 (1953).
[CrossRef] [PubMed]

R. Barer, Nature 172, 1097 (1953).
[CrossRef] [PubMed]

R. Barer, Nature 169, 366 (1952).
[CrossRef] [PubMed]

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

Naturwissenschaften (2)

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

R. Barer, Naturwissenschaften 41, 206 (1954).
[CrossRef]

Pflügers Arch. ges. Physiol. (1)

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

Proc. Phys. Soc. (London) (2)

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

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

Proc. Roy. Soc. (London) (4)

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

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

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

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

Protoplasma (1)

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

Quart. J. Microscop. Sci. (3)

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

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

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

Science (1)

R. Barer, Science 121, 709 (1955).
[CrossRef] [PubMed]

Other (8)

W. D. Wright, Researches on Normal and Defective Colour Vision (Henry Kimpton, London1946).

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

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 and S. Joseph (to be published).

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

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

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Figures (7)

Fig. 1
Fig. 1

Diagram (not to scale) showing refractive indexes of different parts of rods and cones. The figures in brackets are the concentrations of solids corresponding to the refractive indexes given O.S.=outer segment. E=ellipsoid. M=myoid. P=paraboloid. N=nucleus. Average results for different species obtained by Dr. R. L. Sidman.

Fig. 2
Fig. 2

Summary of distribution of hemoglobin concentrations in red cell populations of 500 cases (Miss F. M. Gaffney). See text for explanation.

Fig. 3
Fig. 3

Relationship between concentrations of cytoplasmic solids in fibroblasts and osmotic pressure of medium (Dr. D. A. T. Dick).

Fig. 4
Fig. 4

Effect of changing the refractive index of the immersion medium on the phase-contrast image of a living spherical cell. On the left the cell is mounted in a dilute salt solution and virtually all internal detail is lost. On the right a protein solution of refractive index approximately 1.35 is used, and the internal structure with pale nucleoplasm and dark chromosomes is revealed.

Fig. 5
Fig. 5

The effect of immersion of dead cells in different media. (a) Water. (b) 40% protein. (c) Clove oil. (d) Carbon disulfide. The chromosomes appear bright in (b) and therefore have a refractive index less than 1.40. However, they are almost invisible in (c) and appear to have a refractive index of 1.53.

Fig. 6
Fig. 6

Clarification of cell suspensions. The two cuvettes contain the same concentration of pigmented bacteria, but the suspension on the right is mounted in normal culture medium, that on the left in a protein solution which matches the cells in refractive index. Reproduced by courtesy of Science.27

Fig. 7
Fig. 7

Spectra obtained from clarified suspensions of red-purple photosynthetic bacteria. A and B-Rhodopseudomonas spheroides, grown under different conditions. C-Rhodospirillum rubrum.

Tables (1)

Tables Icon

Table I Specific refraction increments (α) of some substances of biological importance.

Equations (8)

Equations on this page are rendered with MathJax. Learn more.

n = n 0 + α C ,
ϕ = ( n - n M ) t ,
ϕ = α · C · t .
ϕ · A = α · C · A · t .
dry mass = ϕ · A / 100 α
dry mass per unit area = ϕ / 100 α .
ϕ = ( n - n M ) t
ϕ 1 = ( n - n M 1 ) t .