Abstract

A confocal, scanning slit microscope that uses separate portions of the objective aperture for illumination and imaging rays achieves a high degree of optical sectioning. This capability permits visualization of individual cells within the intact inner ear in guinea pigs and cats, and it facilitates directing a laser heterodyne interferometer beam so that vibration of selected cells can be measured. A concentric singlet lens is added to the front of a long-working-distance microscope objective to increase the numerical aperture from 0.4 to 0.53 while retaining a working distance of 6 mm. The measured optical-sectioning capability is compared with the theoretical performance and with the calculated curve for a full-aperture pinhole confocal system.

© 1994 Optical Society of America

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References

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  1. S. M. Khanna, D. G. B. Leonard, “Basilar membrane tuning in the cat cochlea,” Science 215, 305–306 (1982).
    [Crossref] [PubMed]
  2. M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
    [Crossref]
  3. S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
    [Crossref]
  4. J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
    [Crossref]
  5. C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
    [Crossref]
  6. C. J. Koester, “Scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology,” Appl. Opt. 19, 1749–1757 (1980).
    [Crossref] [PubMed]
  7. P. E. Moroz, “A contact cap on common objectives for fluorescence microscopy of living organs,” Microscope 35, 135–144 (1987).
  8. C. J. Koester, J. D. Auran, H. D. Rosskothen, G. J. Florakis, R. B. Tackaberry, “Clinical microscopy of the cornea utilizing optical sectioning and a high numerical aperture objective,” J. Opt. Soc. Am. A 10, 1670–1679 (1993).
    [Crossref] [PubMed]
  9. Model 835 picowatt digital optical power meter with 818-ST detector, Newport Corp., Irvine, Calif.
  10. C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).
  11. T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), p. 72.
  12. M. Petráň, M. Hadravsky, M. D. Egger, R. Galambos, “Tandem-scanning reflected-light microscope,” J. Opt. Soc. Am. 58, 661–664 (1968).
    [Crossref]
  13. W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).
  14. G. S. Kino, “Intermediate optics in Nipkow disk microscopes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 105–111.
    [Crossref]
  15. S. J. Hewlett, T. Wilson, “Optical sectioning in tandem scanning microscopes (TSMs),” Trans. R. Microsc. Soc., 1, 259–262 (1990).
  16. T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 113–126.
    [Crossref]
  17. C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 207–214.
    [Crossref]
  18. Ref. 11, pp. 48–49 and 140–141.

1993 (1)

1990 (3)

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).

S. J. Hewlett, T. Wilson, “Optical sectioning in tandem scanning microscopes (TSMs),” Trans. R. Microsc. Soc., 1, 259–262 (1990).

1989 (4)

M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
[Crossref]

S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
[Crossref]

J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
[Crossref]

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

1987 (1)

P. E. Moroz, “A contact cap on common objectives for fluorescence microscopy of living organs,” Microscope 35, 135–144 (1987).

1982 (1)

S. M. Khanna, D. G. B. Leonard, “Basilar membrane tuning in the cat cochlea,” Science 215, 305–306 (1982).
[Crossref] [PubMed]

1980 (1)

1968 (1)

Auran, J. D.

Cavanagh, H. D.

W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).

Dandliker, R.

J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
[Crossref]

Egger, M. D.

Flock, A.

M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
[Crossref]

Florakis, G. J.

Galambos, R.

Hadravsky, M.

Hewlett, S. J.

S. J. Hewlett, T. Wilson, “Optical sectioning in tandem scanning microscopes (TSMs),” Trans. R. Microsc. Soc., 1, 259–262 (1990).

Jester, J. V.

W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).

Khanna, S. M.

M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
[Crossref]

S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
[Crossref]

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
[Crossref]

S. M. Khanna, D. G. B. Leonard, “Basilar membrane tuning in the cat cochlea,” Science 215, 305–306 (1982).
[Crossref] [PubMed]

Kino, G. S.

G. S. Kino, “Intermediate optics in Nipkow disk microscopes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 105–111.
[Crossref]

Koester, C. J.

C. J. Koester, J. D. Auran, H. D. Rosskothen, G. J. Florakis, R. B. Tackaberry, “Clinical microscopy of the cornea utilizing optical sectioning and a high numerical aperture objective,” J. Opt. Soc. Am. A 10, 1670–1679 (1993).
[Crossref] [PubMed]

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

C. J. Koester, “Scanning mirror microscope with optical sectioning characteristics: applications in ophthalmology,” Appl. Opt. 19, 1749–1757 (1980).
[Crossref] [PubMed]

C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 207–214.
[Crossref]

Leonard, D. G. B.

S. M. Khanna, D. G. B. Leonard, “Basilar membrane tuning in the cat cochlea,” Science 215, 305–306 (1982).
[Crossref] [PubMed]

Moroz, P. E.

P. E. Moroz, “A contact cap on common objectives for fluorescence microscopy of living organs,” Microscope 35, 135–144 (1987).

Petrán, M.

Petroll, W. M.

W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).

Rosskothen, H.

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

Rosskothen, H. D.

Sheppard, C.

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), p. 72.

Tackaberry, R. B.

C. J. Koester, J. D. Auran, H. D. Rosskothen, G. J. Florakis, R. B. Tackaberry, “Clinical microscopy of the cornea utilizing optical sectioning and a high numerical aperture objective,” J. Opt. Soc. Am. A 10, 1670–1679 (1993).
[Crossref] [PubMed]

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

Ulfendahl, M.

M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
[Crossref]

S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
[Crossref]

Willemin, J.-F.

J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
[Crossref]

S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
[Crossref]

Wilson, T.

S. J. Hewlett, T. Wilson, “Optical sectioning in tandem scanning microscopes (TSMs),” Trans. R. Microsc. Soc., 1, 259–262 (1990).

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 113–126.
[Crossref]

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), p. 72.

Acta Oto-Laryngol. (Stockholm) Suppl. (4)

M. Ulfendahl, A. Flock, S. M. Khanna, “Isolated cochlea preparation for the study of cellular vibrations and motility,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 91–96 (1989).
[Crossref]

S. M. Khanna, J.-F. Willemin, M. Ulfendahl, “Measurement of optical reflectivity in cells of the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 69–75 (1989).
[Crossref]

J.-F. Willemin, S. M. Khanna, R. Dandliker, “Heterodyne interferometer for cellular vibration measurement,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 35–42 (1989).
[Crossref]

C. J. Koester, S. M. Khanna, H. Rosskothen, R. B. Tackaberry, “Incident light optical sectioning microscope for visualization of cellular structures in the inner ear,” Acta Oto-Laryngol. (Stockholm) Suppl. 467, 27–33 (1989).
[Crossref]

Appl. Opt. (1)

J. Opt. Soc. Am. (1)

J. Opt. Soc. Am. A (1)

Microscope (1)

P. E. Moroz, “A contact cap on common objectives for fluorescence microscopy of living organs,” Microscope 35, 135–144 (1987).

Science (1)

S. M. Khanna, D. G. B. Leonard, “Basilar membrane tuning in the cat cochlea,” Science 215, 305–306 (1982).
[Crossref] [PubMed]

Trans. R. Microsc. Soc. (3)

W. M. Petroll, H. D. Cavanagh, J. V. Jester, “In vivo digital image acquisition in confocal microscopy,” Trans. R. Microsc. Soc. 1, 349–352 (1990).

S. J. Hewlett, T. Wilson, “Optical sectioning in tandem scanning microscopes (TSMs),” Trans. R. Microsc. Soc., 1, 259–262 (1990).

C. J. Koester, “High efficiency optical sectioning with confocal slits,” Trans. R. Microsc. Soc. 1, 327–332 (1990).

Other (6)

T. Wilson, C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, London, 1984), p. 72.

Model 835 picowatt digital optical power meter with 818-ST detector, Newport Corp., Irvine, Calif.

T. Wilson, “The role of the pinhole in confocal imaging systems,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 113–126.
[Crossref]

C. J. Koester, “A comparison of various optical sectioning methods: the scanning slit confocal microscope,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 207–214.
[Crossref]

Ref. 11, pp. 48–49 and 140–141.

G. S. Kino, “Intermediate optics in Nipkow disk microscopes,” in Handbook of Biological Confocal Microscopy, J. B. Pawley, ed. (Plenum, New York, 1990), pp. 105–111.
[Crossref]

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Figures (9)

Fig. 1
Fig. 1

Schematic diagram of the optical-sectioning microscope. The path of the visible light is shown by the solid line from the arc lamp at L to the image plane at I. L, 150-W xenon arc lamp with built-in reflector; L1, lens to focus arc at slit S1; S1, adjustable slit (width and height); L2, lens to form virtual image of slit S1 at the back focal plane of the microscope objective; M2, three-sided scanning mirror, which oscillates at 1000 Hz, ±2° about an axis perpendicular to the diagram; BS, beam-splitting prisms with high reflectance at the 45° surface for 633-nm light and high transmission for the rest of the visible spectrum; LA, laser beam (dotted-dashed line), part of the laser heterodyne interferometer (not shown); L3, microscope objective; FP, focal plane; D, aperture divider; L4, field lens; S2, slit, which is adjustable in width; L5, lens to collimate light from slit S2; MC, magnification changer, a turret containing Galilean telescopes for increasing or decreasing the magnification; L6, image-forming lens; I, image plane. The image can be viewed by means of an eyepiece, photographed with a 35-mm camera or received by a video camera.

Fig. 2
Fig. 2

Two forms of dipping-cone objectives: (a) Traditional dipping cone shown in front of a long-working-distance objective, 0.35 total NA. The working distance is ~2 mm. (b) New dipping element shown in front of a 20× Olympus objective, NA 0.4. When the dipping element is immersed in water, the total NA increases to 0.53. The working distance is 6 mm.

Fig. 3
Fig. 3

Experimental optical-section curves obtained with a Nikon 20× objective together with the immersed dipping element, NA 0.53. Positive values on the abscissa indicate that the mirror was farther from the objective than the focal plane. For each curve the width of slit S2 was set, then the width of S1 was adjusted so that its image matched the width of S2. The magnification from the focal plane to slit S2 was 26.5 so that at the focal plane the image of the 0.5-mm slit was 0.019 mm wide. The aperture divider (D in Fig. 1) was 2 mm wide, and the objective aperture diameter was 7 mm. Dotted lines represent light levels measured when several possible sources of stray light were removed from the setup, in succession: A, the mirror was removed from its position in front of the objective; B, the water tank was removed; C, slit S2 was blocked.

Fig. 4
Fig. 4

Comparison of one experimental curve (0.5-mm slit width, 2-mm divider) and the calculated optical-section curve (dashed curves) for the same parameters. Calculated width of the slit image at the focal plane, 0.019 mm.

Fig. 5
Fig. 5

Comparison of calculated optical-section curves for a pinhole confocal system (dashed curves) and the divided-aperture confocal slit system. Theory predicts that the pinhole curve approaches the inverse square law at large distances whereas the divided-aperture slit system has a definite cutoff. The horizontal scale has been expanded from that in Fig. 4 because a narrower slit was used for the calculation.

Fig. 6
Fig. 6

Photomicrograph of the inner ear taken in a temporal bone preparation of a guinea pig. In this figure, the focus was at Reissner’s membrane. Bar, 100 μm.

Fig. 7
Fig. 7

Same preparation as in Fig. 6, but the focus was on the Hensen’s cells (H). Note that the image of outer hair cells (OHC) that is visible in Fig. 8 is nearly extinguished by the optical-sectioning effect in this photomicrograph. Bar, 100 μm.

Fig. 8
Fig. 8

Same preparation as in Figs. 6 and 7, with the focus on the outer hair cells (OHC). Some Hensen’s cells are also seen at this level. Bar, 100 μm.

Fig. 9
Fig. 9

Stained section of the organ of Corti showing outer hair cells (OHC), Hensen’s cells (H), and the basilar membrane (BM). A plot of the calculated optical-section curve is superimposed, showing what fraction of the light reflected or scattered from any level within the organ would reach the image plane. The maximum value is at the focal plane (FP). Bar, 10 μm.

Tables (1)

Tables Icon

Table 1 Optical-Section Half-Height h for Two Objectives and Various Slit Widths a

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