Abstract

A microspectrophotometer of exceptional sensitivity has been constructed to record absorption spectra of visual pigments in single cells using minimum light. With this equipment, pigments of individual cones have been recorded in regions of less than 1 μ radius, with minimum bleaching. The instrument is simple and flexible in design, and uses commercially available components throughout. This paper reviews some general design considerations, the character and performance of our equipment, and some of the results obtained with retinal cones.

© 1964 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
    [CrossRef]
  2. B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
    [CrossRef]
  3. P. Liebman. Biophys. J. 2, 161 (1962).
  4. R. A. Smith, Brit. J. Inst. Elect. Engr. 98, 43 (1951).
  5. C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954).
    [CrossRef]
  6. T. G. R. Rawlins, J. Opt. Soc. Am. 54, 423 (1964).
    [CrossRef]
  7. G. P. Brouwer, Phillips Res. Rept. 18, 361 (1963).
  8. J. B. Johnson and R. B. Llewellyn, Bell Sys. Tech. J. 14, 85 (1935).
    [CrossRef]
  9. W. B. Marks, Doctoral dissertation, Johns Hopkins University (1963).

1964 (1)

1963 (1)

G. P. Brouwer, Phillips Res. Rept. 18, 361 (1963).

1962 (1)

P. Liebman. Biophys. J. 2, 161 (1962).

1959 (1)

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

1954 (1)

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954).
[CrossRef]

1951 (1)

R. A. Smith, Brit. J. Inst. Elect. Engr. 98, 43 (1951).

1940 (1)

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[CrossRef]

1935 (1)

J. B. Johnson and R. B. Llewellyn, Bell Sys. Tech. J. 14, 85 (1935).
[CrossRef]

Åkerman, L.

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Brouwer, G. P.

G. P. Brouwer, Phillips Res. Rept. 18, 361 (1963).

Caspersson, T.

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[CrossRef]

Chance, B.

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Johnson, J. B.

J. B. Johnson and R. B. Llewellyn, Bell Sys. Tech. J. 14, 85 (1935).
[CrossRef]

Legallais, V.

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954).
[CrossRef]

Liebman, P.

P. Liebman. Biophys. J. 2, 161 (1962).

Llewellyn, R. B.

J. B. Johnson and R. B. Llewellyn, Bell Sys. Tech. J. 14, 85 (1935).
[CrossRef]

Marks, W. B.

W. B. Marks, Doctoral dissertation, Johns Hopkins University (1963).

Perry, R.

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Rawlins, T. G. R.

Smith, R. A.

R. A. Smith, Brit. J. Inst. Elect. Engr. 98, 43 (1951).

Thorell, B.

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

Yang, C. C.

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954).
[CrossRef]

Bell Sys. Tech. J. (1)

J. B. Johnson and R. B. Llewellyn, Bell Sys. Tech. J. 14, 85 (1935).
[CrossRef]

Biophys. J. (1)

P. Liebman. Biophys. J. 2, 161 (1962).

Brit. J. Inst. Elect. Engr. (1)

R. A. Smith, Brit. J. Inst. Elect. Engr. 98, 43 (1951).

J. Opt. Soc. Am. (1)

J. Roy. Microscop. Soc. (1)

T. Caspersson, J. Roy. Microscop. Soc. 60, 8 (1940).
[CrossRef]

Phillips Res. Rept. (1)

G. P. Brouwer, Phillips Res. Rept. 18, 361 (1963).

Rev. Sci. Instr. (2)

B. Chance, R. Perry, L. Åkerman, and B. Thorell, Rev. Sci. Instr. 30, 735 (1959).
[CrossRef]

C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954).
[CrossRef]

Other (1)

W. B. Marks, Doctoral dissertation, Johns Hopkins University (1963).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (12)

F. 1
F. 1

Block diagram of apparatus.

F. 2
F. 2

Upper part of the figure illustrates the light and signal voltage pulses during one chopping cycle. The reference pulse is made larger than the sample pulse to diminish reference-channel noise. The lower diagrams show the operation of the demodulating switch.

F. 3
F. 3

Light chopping disc shown superimposed on aperture plate containing two circular apertures A. The disc is driven by a 30 cps synchronous motor to allow light to pass through one hole at a time.

F. 4
F. 4

Optical systems tested in this work. A shows Koehler illumination with a grating monochromator. B shows critical illumination. C shows running-interference-filter monochromator illumination which provides a type of Koehler illumination free of monochromator images.

F. 5
F. 5

A baseline anomaly at about 605 mμ is seen in the upper tracing. When a linear polarizer is inserted into the light beam at the exit slit, this anomaly can be decomposed into two components, only one of which retains the anomaly (I and II).

F. 6
F. 6

Electronic circuit.

F. 7
F. 7

Test of response speed of microspectrophotometer with Wratten 58 filter. (A) true spectrum—1 mμ/sec. (B): fast scan without distortion—15 mμ/sec, 0.01 sec time constant. (C): distortion due to slower response time at high scanning rate—15 mμ/sec, 0.15 sec time constant.

F. 8
F. 8

Noise level as function of light intensity. Neutral density filters (D0.6 and D0.9) were inserted in the light beam. The increase in noise is that expected for photoelectron shot-noise-limited performance.

F. 9
F. 9

Two successive absorption spectra recorded from a 1.9 mm circular region of a single goldfish cone showing that insignificant bleaching has occurred in the measurement.

F. 10
F. 10

Absorption spectra obtained from three different cones from the goldfish. These are all free of bleaching distortion.

F. 11
F. 11

Bleaching kinetics of a cone containing the 537 mμ pigment. Time proceeds from right to left. This shows the pigment is photosensitive, decaying by a first-order reaction.

F. 12
F. 12

Absorption spectrum from mitochondria-containing region of cell. The proportions of the different cytochromes can easily be determined from this record.