Abstract

We present a quantitative phase-contrast confocal microscope (QPCCM) by combining a line-scanning confocal system with digital holography (DH). This combination can merge the merits of these two different imaging modalities. High-contrast intensity images with low coherent noise, and the optical sectioning capability are made available due to the confocality. Phase profiles of the samples become accessible thanks to DH. QPCCM is able to quantitatively measure the phase variations of optical sections of the opaque samples and has the potential to take high-quality intensity and phase images of non-opaque samples such as many biological samples. Because each line scan is recorded by a hologram that may contain the optical aberrations of the system, it opens avenues for a variety of numerical aberration compensation methods and development of full digital adaptive optics confocal system to emulate current hardware-based adaptive optics system for biomedical imaging, especially ophthalmic imaging. Preliminary experiments with a microscope objective of NA 0.65 and 40 × on opaque samples are presented to demonstrate this idea. The measured lateral and axial resolutions of the intensity images from the current system are ~0.64μm and ~2.70μm respectively. The noise level of the phase profile by QPCCM is ~2.4nm which is better than the result by DH.

© 2014 Optical Society of America

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References

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2013 (1)

2012 (2)

2011 (1)

2010 (1)

M. K. Kim, “Principles and techniques of digital holographic microscopy,” SPIE Reviews 1, 1–50 (2010).

2009 (1)

2008 (2)

2007 (1)

2006 (2)

P. J. Dwyer, C. A. DiMarzio, J. M. Zavislan, W. J. Fox, and M. Rajadhyaksha, “Confocal reflectance theta line scanning microscope for imaging human skin in vivo,” Opt. Lett. 31(7), 942–944 (2006).
[CrossRef] [PubMed]

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

2005 (2)

2003 (1)

1999 (1)

1996 (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[CrossRef]

1987 (1)

Bevilacqua, F.

Bigelow, C. E.

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

Carlini, A. R.

Cuche, E.

Depeursinge, C.

DiMarzio, C. A.

Dwyer, P. J.

Ferguson, R. D.

M. Mujat, R. D. Ferguson, N. Iftimia, and D. X. Hammer, “Compact adaptive optics line scanning ophthalmoscope,” Opt. Express 17(12), 10242–10258 (2009).
[CrossRef] [PubMed]

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

Fienup, J. R.

Fox, W. J.

Goy, A. S.

Hammer, D. X.

M. Mujat, R. D. Ferguson, N. Iftimia, and D. X. Hammer, “Compact adaptive optics line scanning ophthalmoscope,” Opt. Express 17(12), 10242–10258 (2009).
[CrossRef] [PubMed]

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

Han, S.

Iftimia, N.

Iftimia, N. V.

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

Im, K. B.

Khmaladze, A.

Kim, B. M.

Kim, D.

Kim, M. K.

Liu, C.

Lo, C. M.

Mann, C.

Miller, J. J.

Mujat, M.

Park, H.

Psaltis, D.

Rajadhyaksha, M.

Thurman, S. T.

Unser, M.

Ustun, T. E.

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

Webb, R. H.

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[CrossRef]

Wilson, T.

Yu, L.

Yu, X.

Zavislan, J. M.

Appl. Opt. (2)

Biomed. Opt. Express (1)

J. Biomed. Opt. (1)

D. X. Hammer, R. D. Ferguson, T. E. Ustun, C. E. Bigelow, N. V. Iftimia, and R. H. Webb, “Line-scanning laser ophthalmoscope,” J. Biomed. Opt. 11(4), 041126 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (2)

Opt. Express (5)

Opt. Lett. (4)

Rep. Prog. Phys. (1)

R. H. Webb, “Confocal optical microscopy,” Rep. Prog. Phys. 59(3), 427–471 (1996).
[CrossRef]

SPIE Reviews (1)

M. K. Kim, “Principles and techniques of digital holographic microscopy,” SPIE Reviews 1, 1–50 (2010).

Other (4)

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

T. Wilson, ed., Confocal Microscopy (Academic, 1990).

T. R. Corle and G. S. Kino, eds., Confocal Scanning Optical Microscopy and Related Imaging Systems (Academic, 1996).

M. Minsky, “Microscopy apparatus,” U.S. patent 3,013,467 (December 1961).

Supplementary Material (1)

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Figures (6)

Fig. 1
Fig. 1

Schematic diagram of the optical system. (a) Top view of the setup. B1-B3: Cubic beam splitters, B4-B6: Pellicles, BE1-BE3: Beam expanders, L1-L2: Spherical lens. MO: Microscope objective. CL: Cylindrical lens. M: Mirror. (b) View of the xz plane of the illumination. (c) View of the yz plane of the illumination.

Fig. 2
Fig. 2

Reconstructions of confocal intensity image and confocal phase map. (a) Hologram of one line scan. All the scans are contained in the video Media 1. (b) Detailed view of the region in the white square in (a). (c) Angular spectrum. (d) Reconstructed intensity of the line scan. The green rectangle represents the numerical slit. (e) Reconstructed phase of the line scan. (f) Confocal intensity image. (g) Wide-field image by He-Ne laser illumination. (h) Confocal phase map (in radian). (i) Corrected phase map. Scale bar in (g):10μm. (f), (g) and (i) have same field of view.

Fig. 3
Fig. 3

Measurements of lateral and axial resolutions. (a) Edge spread function in x direction. (b) Edge spread function in y direction. (c) Axial response with respect to the axial distance away from the focal plane.

Fig. 4
Fig. 4

Phase images of a phase object by QPCCM and DH. (a) Phase map by QPCCM. (b) Height profile of a cross section by the solid line in (a). (c) Height profile of a cross section by the dashed line in (a). (d) Phase map by DH. (e) Height profile of a cross section indicated by the solid line in (d).(f) Height profile of a cross section indicated by the dashed line in (d). (g) Three-dimensional pseudo-color rendering of (a). (h) Three-dimensional pseudo-color rendering of (d). Fields of view of (a) and (d) are 37.4 × 37.4μm2.

Fig. 5
Fig. 5

The effect of slit width on the phase profile. (a) The phase profile versus the slit width. (b) The measured height versus the slit width.

Fig. 6
Fig. 6

Confocal intensity images and phase maps of optical sections of a silicon wafer. (a)-(c) Wide-field images at z = 0μm,10μm, and 20μm. (d)-(f) Confocal intensity images at z = 0μm,10μm, and 20μm. (g) Confocal xz section at the position in xy plane indicated by the dashed line in (f). (h)-(j) Scanning images without numerical slit at z = 0μm,10μm, and 20μm. (k) Non-confocal counterpart of (g). (l)-(n) Confocal phase maps at z = 0μm,10μm, and 20μm. Scale bars in (a) and (g):10μm.

Equations (5)

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I conf (x,n)= yslit I n (x,y)
S w = 0.61λM P×NA
Φ conf (x,n)= yslit Φ n (x,y) S w
Height= Phase 4π λ
A.U.= 1.22λ NA

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