Abstract

We demonstrate a simple phase-sensitive microscopic technique capable of imaging the phase gradient of a transparent specimen, based on the Schlieren modulation and confocal laser scanning microscopy (CLSM). The incident laser is refracted by the phase gradient of the specimen and excites a fluorescence plate behind the specimen to create a secondary illumination; then the fluoresence is modulated by a partial obstructor before entering the confocal pinhole. The quantitative relationship between the image intensity and the sample phase gradient can be derived. This setup is very easy to be adapted to current confocal setups, so that multimodality fluorescence/structure images can be obtained within a single system.

© 2014 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. F. Zernike, Physica 9, 686 (1942).
    [CrossRef]
  2. G. Nomarski, J. Phys. Radium 16, 9S (1955).
  3. C. Preza, D. L. Snyder, and J.-A. Conchello, J. Opt. Soc. Am. A 16, 2185 (1999).
    [CrossRef]
  4. C. J. Cogswell and C. Sheppard, J. Microsc. 165, 81 (1992).
    [CrossRef]
  5. G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, Opt. Lett. 31, 775 (2006).
    [CrossRef]
  6. S. Fürhapter, A. Jesacher, S. Bernet, and M. Ritsch-Marte, Opt. Express 13, 689 (2005).
    [CrossRef]
  7. M. Davidson and M. Abramowitz, in Encyclopedia of Imaging Science and Technology, J. Honak, ed. (Wiley, 2002).
  8. J. G. Dodd, Appl. Opt. 16, 470 (1977).
    [CrossRef]
  9. D. Axelrod, Cell Biophys. 3, 167 (1981).
  10. R. Yi, K. K. Chu, and J. Mertz, Opt. Express 14, 5191 (2006).
    [CrossRef]
  11. R. Hoffman and L. Gross, Appl. Opt. 14, 1169 (1975).
    [CrossRef]
  12. J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 1995).
  13. Y. Ding, H. Xie, T. Peng, Y. Lu, D. Jin, J. Teng, Q. Ren, and P. Xi, Opt. Express 20, 14100 (2012).
    [CrossRef]
  14. M. Born, E. Wolf, and A. Bhatia, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University, 1999).
  15. T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984), Chap. 3, pp. 37–42.
  16. J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
    [CrossRef]

2012 (1)

2006 (2)

2005 (1)

1999 (1)

1996 (1)

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

1992 (1)

C. J. Cogswell and C. Sheppard, J. Microsc. 165, 81 (1992).
[CrossRef]

1981 (1)

D. Axelrod, Cell Biophys. 3, 167 (1981).

1977 (1)

1975 (1)

1955 (1)

G. Nomarski, J. Phys. Radium 16, 9S (1955).

1942 (1)

F. Zernike, Physica 9, 686 (1942).
[CrossRef]

Abramowitz, M.

M. Davidson and M. Abramowitz, in Encyclopedia of Imaging Science and Technology, J. Honak, ed. (Wiley, 2002).

Axelrod, D.

D. Axelrod, Cell Biophys. 3, 167 (1981).

Bernet, S.

Beuthan, J.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Bhatia, A.

M. Born, E. Wolf, and A. Bhatia, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University, 1999).

Born, M.

M. Born, E. Wolf, and A. Bhatia, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University, 1999).

Chu, K. K.

Cogswell, C. J.

C. J. Cogswell and C. Sheppard, J. Microsc. 165, 81 (1992).
[CrossRef]

Conchello, J.-A.

Dasari, R. R.

Davidson, M.

M. Davidson and M. Abramowitz, in Encyclopedia of Imaging Science and Technology, J. Honak, ed. (Wiley, 2002).

Ding, Y.

Dodd, J. G.

Feld, M. S.

Fürhapter, S.

Gross, L.

Helfmann, J.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Herrig, M.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Hoffman, R.

Ikeda, T.

Jesacher, A.

Jin, D.

Lu, Y.

Mertz, J.

Minet, O.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Müller, G.

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Nomarski, G.

G. Nomarski, J. Phys. Radium 16, 9S (1955).

Pawley, J. B.

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 1995).

Peng, T.

Popescu, G.

Preza, C.

Ren, Q.

Ritsch-Marte, M.

Sheppard, C.

C. J. Cogswell and C. Sheppard, J. Microsc. 165, 81 (1992).
[CrossRef]

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984), Chap. 3, pp. 37–42.

Snyder, D. L.

Teng, J.

Wilson, T.

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984), Chap. 3, pp. 37–42.

Wolf, E.

M. Born, E. Wolf, and A. Bhatia, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University, 1999).

Xi, P.

Xie, H.

Yi, R.

Zernike, F.

F. Zernike, Physica 9, 686 (1942).
[CrossRef]

Appl. Opt. (2)

Cell Biophys. (1)

D. Axelrod, Cell Biophys. 3, 167 (1981).

J. Microsc. (1)

C. J. Cogswell and C. Sheppard, J. Microsc. 165, 81 (1992).
[CrossRef]

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

J. Phys. Radium (1)

G. Nomarski, J. Phys. Radium 16, 9S (1955).

Opt. Express (3)

Opt. Lett. (1)

Phys. Med. Biol. (1)

J. Beuthan, O. Minet, J. Helfmann, M. Herrig, and G. Müller, Phys. Med. Biol. 41, 369 (1996).
[CrossRef]

Physica (1)

F. Zernike, Physica 9, 686 (1942).
[CrossRef]

Other (4)

M. Davidson and M. Abramowitz, in Encyclopedia of Imaging Science and Technology, J. Honak, ed. (Wiley, 2002).

J. B. Pawley, Handbook of Biological Confocal Microscopy (Springer, 1995).

M. Born, E. Wolf, and A. Bhatia, Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge University, 1999).

T. Wilson and C. Sheppard, Theory and Practice of Scanning Optical Microscopy (Academic, 1984), Chap. 3, pp. 37–42.

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 (4)

Fig. 1.
Fig. 1.

Schematic diagram of S-confocal. (a) S-confocal is based on a CLSM, except for a fluorescent plate placed behind the specimen, and a block in front of the fluorescence collection. FM, fluorescent medium; PO, phase object; CP, confocal pinhole; Obj, objective lens; TL, tube lens; SL, scan lens; Obs, obstructor; DM, dichromic mirror; galva, galvonometer. Fluorescence illuminates the whole sample, but only the optical rays that go through the neighborhood of the scanning point could be detected. (b) The phase gradient of the specimen acts as a micro-prism to effectively deflect the incident/fluorescent beams. Here, only the incident laser beam is drawn. (c) Coordinates on the sample scanning stage. O is the laboratory frame, and O is a moving frame on the sample. The coordination of the scanning point in the sample frame is xs.

Fig. 2.
Fig. 2.

(a) Laser scanning S-confocal system to illustrate the effect of incident and detection gradients. (b)–(d) show S-confocal image with UV laser incident, fluorescence medium on top and pinhole of 10 AU. The obstruction direction determines the orientation of the shade effect. Image intensity indicates the incident gradient, since the detection gradient is averaged. (e)–(g) are scanning images with Köehler illumination with a 3 AU pinhole, but without laser and fluorescence medium. (h)–(j) show the S-confocal image with UV laser incident, fluorescence medium on top and pinhole of 3 AU. Thereby, the intensity in (h)–(j) is contributed by both the incident gradient in (b)–(d), and the detection gradient in (e)–(g). Left, middle, and right columns are images without obstruction, upside obstruction and downside obstruction, respectively. The image size is 120μm×120μm. (k) Grating profile (green dash and dots), grating gradient (red dashes), and measured gradient (blue solid) along yellow line in Fig. 2(c).

Fig. 3.
Fig. 3.

(a) Fluorescence image of mouse kidney (DAPI stained). (b) Phase-relief image of mouse kidney by S-confocal, and (c) merged image. The image size is 180μm×180μm.

Fig. 4.
Fig. 4.

Type 2 S-confocal image with pinhole of 10 AU and a half-fluorescence plate. (a), (b), and (c) are images with one full fluorescence plate and two half-fluorescence plates with different directions, respectively. The image size is 60μm×60μm.

Equations (5)

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

U(x0,xs)=U0+h(x1)t(x1+xs)expikx0x1ddx1,
If(x0,xs)=If0ξ(g(xs)+x0/da/f).
Idet(xs,xd)=If(x0,xs)η(g(xd)dx0)dx0,
Idet(xs,xd)=If(x0,xs)η(g¯(xd)x0)dx0,
Idet(xs)=I0(1+1ka/fdϕ(xs)dxs).

Metrics