Abstract

We present a high-speed and phase-sensitive reflectance line-scanning confocal holographic microscope (LCHM). We achieved rapid confocal imaging using a fast line-scan CCD camera and quantitative phase imaging using off-axis digital holography (DH) on a 1D, line-by-line basis in our prototype experiment. Using a 20 kHz line scan rate, we achieved a frame rate of 20 Hz for 512x512 pixels en-face confocal images. We realized coherent holographic detection two different ways. We first present a LCHM using off-axis configuration. By using a microscope objective of a NA 0.65, we achieved axial and lateral resolution of ~3.5 micrometers and ~0.8 micrometers, respectively. We demonstrated surface profile measurement of a phase target at nanometer precision and the digital refocusing of a defocused confocal en-face image. Ultrahigh temporal resolution M mode is demonstrated by measuring the vibration of a PZT-actuated mirror driven by a sine wave at 1 kHz. We then report our experimental work on a LCHM using an in-line configuration. In this in-line LCHM, the coherent detection is enabled by moving the reference arm at a constant speed, thereby introducing a Doppler frequency shift that leads to spatial interference fringes along the scanning direction. Lastly, we present a unified formulation that treats off-axis and in-line LCHM in a unified joint spatiotemporal modulation framework and provide a connection between LCHM and the traditional off-axis DH. The presented high-speed LCHM may find applications in optical metrology and biomedical imaging.

© 2016 Optical Society of America

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References

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    [Crossref]

2015 (1)

C. Liu and M. K. Kim, “Digital adaptive optics line-scanning confocal imaging system,” J. Biomed. Opt. 20(11), 111203 (2015).
[Crossref] [PubMed]

2014 (4)

2013 (1)

2012 (2)

A. S. Goy and D. Psaltis, “Digital confocal microscope,” Opt. Express 20(20), 22720–22727 (2012).
[Crossref] [PubMed]

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

2010 (2)

2009 (1)

2008 (1)

2007 (1)

R. K. Wang, “In-vivo full range complex Fourier domain optical coherence tomography,” Appl. Phys. Lett. 90(5), 054103 (2007).
[Crossref] [PubMed]

2006 (1)

2005 (2)

K. B. Im, S. Han, H. Park, D. Kim, and B. M. Kim, “Simple high-speed confocal line-scanning microscope,” Opt. Express 13(13), 5151–5156 (2005).
[Crossref] [PubMed]

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

2000 (1)

M. G. Somekh, C. W. See, and J. Goh, “Wide field amplitude and phase confocal microscope with speckle illumination,” Opt. Commun. 174(1-4), 75–80 (2000).
[Crossref]

1999 (1)

1997 (1)

R. A. Herring, “Confocal scanning laser holography, and an associated microscope: a proposal,” Optik (Stuttg.) 105, 65–68 (1997).

1996 (1)

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

1988 (1)

M. Minsky, “Memoir on inventing scanning confocal microscope,” Scanning 10(4), 128–138 (1988).
[Crossref]

Adie, S. G.

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Ahmad, A.

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Amthor, F.

Bevilacqua, F.

Boppart, S. A.

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Bromberg, Y.

Cao, H.

Carney, P. S.

M. Schnell, M. J. Perez-Roldan, P. S. Carney, and R. Hillenbrand, “Quantitative confocal phase imaging by synthetic optical holography,” Opt. Express 22(12), 15267–15276 (2014).
[Crossref] [PubMed]

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Choi, W.

Choi, Y.

Choma, M. A.

Cuche, E.

Dasari, R. R.

Depeursinge, C.

DiMarzio, C. A.

Dwyer, P. J.

Ferguson, R. D.

Fienup, J. R.

Fox, W. J.

Goh, J.

M. G. Somekh, C. W. See, and J. Goh, “Wide field amplitude and phase confocal microscope with speckle illumination,” Opt. Commun. 174(1-4), 75–80 (2000).
[Crossref]

Goy, A. S.

Graf, B. W.

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Hammer, D. X.

Han, S.

Herring, R. A.

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

R. A. Herring, “Confocal scanning laser holography, and an associated microscope: a proposal,” Optik (Stuttg.) 105, 65–68 (1997).

Hillenbrand, R.

Hosseini, P.

Iftimia, N.

Im, K. B.

Jacquemin, P.

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

Kim, B. M.

Kim, D.

Kim, M. K.

C. Liu and M. K. Kim, “Digital adaptive optics line-scanning confocal imaging system,” J. Biomed. Opt. 20(11), 111203 (2015).
[Crossref] [PubMed]

C. Liu, S. Marchesini, and M. K. Kim, “Quantitative phase-contrast confocal microscope,” Opt. Express 22(15), 17830–17839 (2014).
[Crossref] [PubMed]

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

Lai, S.

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

Laurin, D.

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

Li, Y. G.

Liu, C.

C. Liu and M. K. Kim, “Digital adaptive optics line-scanning confocal imaging system,” J. Biomed. Opt. 20(11), 111203 (2015).
[Crossref] [PubMed]

C. Liu, S. Marchesini, and M. K. Kim, “Quantitative phase-contrast confocal microscope,” Opt. Express 22(15), 17830–17839 (2014).
[Crossref] [PubMed]

Liu, L.

Marchesini, S.

McLeod, R.

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

Minsky, M.

M. Minsky, “Memoir on inventing scanning confocal microscope,” Scanning 10(4), 128–138 (1988).
[Crossref]

Mujat, M.

Park, H.

Perez-Roldan, M. J.

Psaltis, D.

Rajadhyaksha, M.

Redding, B.

Schnell, M.

See, C. W.

M. G. Somekh, C. W. See, and J. Goh, “Wide field amplitude and phase confocal microscope with speckle illumination,” Opt. Commun. 174(1-4), 75–80 (2000).
[Crossref]

So, P. T. C.

Somekh, M. G.

M. G. Somekh, C. W. See, and J. Goh, “Wide field amplitude and phase confocal microscope with speckle illumination,” Opt. Commun. 174(1-4), 75–80 (2000).
[Crossref]

Thurman, S. T.

Unser, M.

Wang, R. K.

R. K. Wang, “In-vivo full range complex Fourier domain optical coherence tomography,” Appl. Phys. Lett. 90(5), 054103 (2007).
[Crossref] [PubMed]

Webb, R. H.

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

Yao, X. C.

Yaqoob, Z.

Zavislan, J. M.

Appl. Phys. Lett. (1)

R. K. Wang, “In-vivo full range complex Fourier domain optical coherence tomography,” Appl. Phys. Lett. 90(5), 054103 (2007).
[Crossref] [PubMed]

Biomed. Opt. Express (1)

J. Biomed. Opt. (1)

C. Liu and M. K. Kim, “Digital adaptive optics line-scanning confocal imaging system,” J. Biomed. Opt. 20(11), 111203 (2015).
[Crossref] [PubMed]

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

Microgravity Sci. Technol. (1)

P. Jacquemin, R. McLeod, D. Laurin, S. Lai, and R. A. Herring, “Design of a confocal holography microscope for Three-dimensional temperature measurements of fluids in microgravity,” Microgravity Sci. Technol. 17(4), 36–40 (2005).
[Crossref]

Opt. Commun. (1)

M. G. Somekh, C. W. See, and J. Goh, “Wide field amplitude and phase confocal microscope with speckle illumination,” Opt. Commun. 174(1-4), 75–80 (2000).
[Crossref]

Opt. Express (5)

Opt. Lett. (5)

Optik (Stuttg.) (1)

R. A. Herring, “Confocal scanning laser holography, and an associated microscope: a proposal,” Optik (Stuttg.) 105, 65–68 (1997).

Proc. Natl. Acad. Sci. U.S.A. (1)

S. G. Adie, B. W. Graf, A. Ahmad, P. S. Carney, and S. A. Boppart, “Computational adaptive optics for broadband optical interferometric tomography of biological tissue,” Proc. Natl. Acad. Sci. U.S.A. 109(19), 7175–7180 (2012).
[Crossref] [PubMed]

Rep. Prog. Phys. (1)

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

Scanning (1)

M. Minsky, “Memoir on inventing scanning confocal microscope,” Scanning 10(4), 128–138 (1988).
[Crossref]

SPIE Reviews (1)

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

Other (4)

R. N. Bryan, Introduction to the Science of Medical Imaging (Cambridge University Press, 2009).

L. Mastropasqua and M. Nubile, Confocal Microscopy of the Cornea (Slack Incorporated, 2002).

J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (Springer, 2006), pp. 381–439.

T. R. Corle and G. S. Kino, eds., Confocal Scanning Optical Microscopy and Related Imaging Systems (Academic, 1996), pp. 225–275.

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Figures (8)

Fig. 1
Fig. 1 Schematic diagram of optical apparatus of the off-axis LCHM. (a) Top view (x-z plane or the optical tabletop plane) of the optical layout. CL: cylindrical lens with a focal length of 100mm. GSM: Galvanometer scanning mirror. MO1 and MO2: Microscope objectives (NA 0.65, 65 × ). L1-L3: regular singlet lens. Focal lengths are 300mm, 35mm, and 125mm respectively. (b) Illumination path at x-z plane. (c) Illumination path at y-z plane.
Fig. 2
Fig. 2 Demonstration of the principle of operation and data processing. (a) Digital hologram of 512 line holograms. (b) Magnified view of the region in (a) highlighted by the green square. (c) AS of (a) by taking 1D FT of (a) along y direction. (d) Profile along the dashed white line in (c). (e) Reconstructed intensity. (f) Profile along dashed white line in (e). (g) Reconstructed phase map (in radians) from the hologram (a). (h) The corrected phase map.
Fig. 3
Fig. 3 Quantitative phase imaging of a phase target. (a) Raw hologram. (b) Magnified view of the region in (a) indicated by the green square. (c) The surface height map of the phase target after correction. (d) The profile along solid black line in (c). (e) 3D rendering of (c).
Fig. 4
Fig. 4 Axial resolution measurement and experimental demonstration using a silicon wafer. (a) Axial responses with respect to the distance away from the focal plane from LCHM and LCM. (b) En-face intensity image at the top layer from LCHM. (c) En-face phase map at the top layer from LCHM. (d) En-face intensity image at the bottom layer, and (e) En-face phase map at the top layer. (f) y-z section along the x position indicated by the dashed line in (d). Scale bars: 10 μm.
Fig. 5
Fig. 5 Demonstration of digital refocusing by LCHM. (a), and (b) are the intensity and phase of the defocused field respectively. (c), and (d) are the refocused fields by numerically propagating the field of (a) and (b) by a distance of 110mm in image space.
Fig. 6
Fig. 6 Vibration measurement by LCHM. (a) Hologram at y-t plane at V = 0 Volts (Inset × 2). (b) z(y, t) from (a). (c) Hologram at V = 2.5 Volts. (d) z(y, t) from (b). (e) Hologram at V = 5 Volts. (f) z(y, t) from (e).
Fig. 7
Fig. 7 Demonstration of in line LCHM. (a) hologram. (b) magnified view of the region bounded by the green square in (a). (c) AS. (d) Profile along white dashed line in (c). (e) Reconstructed intensity. (f) profile along the white dashed line in (e). (g) original phase map. (h) corrected phase map. (i) axial response with respect to the distance away from the focal plane.
Fig. 8
Fig. 8 Demonstration of joint spatiotemporal phase modulation. (a) hologram from off-axis LCHM when the reference beam is static (Inset 3 × ). (b) 2D FT of (a) or 2D AS of (a). (c) Reconstructed intensity from (a). (d) Hologram when reference arm moves at a speed of ~421μm/s. (e) AS. (f) Reconstructed intensity. (g) Hologram when reference arm moves at a speed of ~633μm/s. (h) AS. (i) Reconstructed intensity. (j): Magnified view of the region within the green square in (c). (k): Magnified view of the region within the green square in (f). (l): Magnified view of the region within the green square in (i).

Equations (7)

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H(x,y)= | O(x,y) | 2 + | R(x,y) | 2 +2| O(x,y) || R(x,y) |cos[2π f yc y+ϕ(x,y)+ ϕ a (x,y)]
H(t,y)= | R(y)exp(j2π f Tc t)+O(t,y) | 2 = | R(y) | 2 + | O(t,y) | 2 +2| R(y) || O(t,y) |cos[2π f Tc t+φ(t,y)+ φ b (t,y)]
f Tc = 2 v R c f 0
x= v x t
H(x,y)= | R(y)exp(j2π f xc x)+O(x,y) | 2 = | R(y) | 2 + | O(x,y) | 2 +2| R(y) || O(x,y) |cos[2π f xc x+φ(x,y)+ φ b (x,y)]
f xc = f Tc v x = 2 v R v x c f 0
H(x,y)= | R(x,y)exp(j2π f xc x)+O(x,y) | 2 = | R(x,y) | 2 + | O(x,y) | 2 +2| R(x,y) || O(x,y) |cos[2π f xc x+2π f yc y+φ(x,y)+ φ c (x,y)]

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