Abstract

We demonstrate quantitative phase mapping in confocal optical microscopy by applying synthetic optical holography (SOH), a recently introduced method for technically simple and fast phase imaging in scanning optical microscopy. SOH is implemented in a confocal microscope by simply adding a linearly moving reference mirror to the microscope setup, which generates a synthetic reference wave analogous to the plane reference wave of wide-field off-axis holography. We demonstrate that SOH confocal microscopy allows for non-contact surface profiling with sub-nanometer depth resolution. As an application for biological imaging, we apply SOH confocal microscopy to map the surface profile of an onion cell, revealing nanoscale-height features on the cell surface.

© 2014 Optical Society of America

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References

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2014 (2)

M. Schnell, P. S. Carney, and R. Hillenbrand, “Synthetic optical holography for rapid nanoimaging,” Nat. Commun. 5, 3499 (2014).
[Crossref] [PubMed]

M. Guillon and M. A. Lauterbach, “Quantitative confocal spiral phase contrast,” J. Opt. Soc. Am. A 31(6), 1215–1225 (2014).
[Crossref]

2012 (1)

2010 (4)

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

B. Deutsch, R. Beams, and L. Novotny, “Nanoparticle detection using dual-phase interferometry,” Appl. Opt. 49(26), 4921–4925 (2010).
[Crossref] [PubMed]

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

2007 (2)

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

N. Warnasooriya and M. K. Kim, “LED-based multi-wavelength phase imaging interference microscopy,” Opt. Express 15(15), 9239–9247 (2007).
[Crossref] [PubMed]

2006 (1)

2002 (1)

2001 (1)

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

2000 (1)

1997 (1)

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

1996 (1)

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

1995 (1)

1986 (2)

1981 (1)

1973 (1)

Abdelwahab, M.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Appel, R. K.

Atalick, S.

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Azhagurajan, M.

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

Barber, B. P.

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

Beams, R.

Carney, P. S.

M. Schnell, P. S. Carney, and R. Hillenbrand, “Synthetic optical holography for rapid nanoimaging,” Nat. Commun. 5, 3499 (2014).
[Crossref] [PubMed]

Coons, S. W.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Delaney, P. M.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Deutsch, B.

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

B. Deutsch, R. Beams, and L. Novotny, “Nanoparticle detection using dual-phase interferometry,” Appl. Opt. 49(26), 4921–4925 (2010).
[Crossref] [PubMed]

Dykes, C.

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

Ek, L.

Eschbacher, J.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Fercher, A. F.

Gammel, P. L.

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

Gopani, S.

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

Götzinger, E.

Goy, A. S.

Graebner, J. E.

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

Greywall, D. S.

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

Guillon, M.

Hamilton, D. K.

Herring, R.

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Herring, R. A.

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

Hillenbrand, R.

M. Schnell, P. S. Carney, and R. Hillenbrand, “Synthetic optical holography for rapid nanoimaging,” Nat. Commun. 5, 3499 (2014).
[Crossref] [PubMed]

Hitzenberger, C. K.

Ignatovich, F.

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

Itaya, K.

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

Jacquemin, P.

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Kim, M. K.

Knuuttila, J. V.

Kobayashi, S.-i.

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

Lahiri, A.

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

Lai, S.

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Lauterbach, M. A.

Matthews, H. J.

McLeod, R. A.

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Mitra, A.

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

Nakaji, P.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Novotny, L.

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

B. Deutsch, R. Beams, and L. Novotny, “Nanoparticle detection using dual-phase interferometry,” Appl. Opt. 49(26), 4921–4925 (2010).
[Crossref] [PubMed]

Pantzer, D.

Parshall, D.

Pircher, M.

Politch, J.

Preul, M. C.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Psaltis, D.

Ryan, R. W.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Salomaa, M. M.

Sankar, T.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Sattmann, H.

Sawatari, T.

Scheck, A. C.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Schnell, M.

M. Schnell, P. S. Carney, and R. Hillenbrand, “Synthetic optical holography for rapid nanoimaging,” Nat. Commun. 5, 3499 (2014).
[Crossref] [PubMed]

Sheppard, C. J. R.

Smith, K. A.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Somekh, M. G.

Sommargren, G. E.

Spetzler, R. F.

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Sticker, M.

Tikka, P. T.

Valera, M. S.

Warnasooriya, N.

Webb, R. H.

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

Wen, R.

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

ACS Nano (1)

A. Mitra, B. Deutsch, F. Ignatovich, C. Dykes, and L. Novotny, “Nano-optofluidic detection of single viruses and nanoparticles,” ACS Nano 4(3), 1305–1312 (2010).
[Crossref] [PubMed]

Appl. Opt. (7)

Appl. Phys. Lett. (1)

J. E. Graebner, B. P. Barber, P. L. Gammel, D. S. Greywall, and S. Gopani, “Dynamic visualization of subangstrom high-frequency surface vibrations,” Appl. Phys. Lett. 78(2), 159–161 (2001).
[Crossref]

J. Am. Chem. Soc. (1)

R. Wen, A. Lahiri, M. Azhagurajan, S.-i. Kobayashi, and K. Itaya, “A new in situ optical microscope with single atomic layer resolution for observation of electrochemical dissolution of Au(111),” J. Am. Chem. Soc. 132(39), 13657–13659 (2010).
[Crossref] [PubMed]

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

Nat. Commun. (1)

M. Schnell, P. S. Carney, and R. Hillenbrand, “Synthetic optical holography for rapid nanoimaging,” Nat. Commun. 5, 3499 (2014).
[Crossref] [PubMed]

Neurosurgery (1)

T. Sankar, P. M. Delaney, R. W. Ryan, J. Eschbacher, M. Abdelwahab, P. Nakaji, S. W. Coons, A. C. Scheck, K. A. Smith, R. F. Spetzler, and M. C. Preul, “Miniaturized handheld confocal microscopy for neurosurgery: results in an experimental glioblastoma model,” Neurosurgery 66(2), 410–418 (2010).
[Crossref] [PubMed]

Opt. Express (2)

Opt. Lett. (2)

Optik (1)

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

Rep. Prog. Phys. (1)

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

Ultramicroscopy (1)

S. Lai, R. A. McLeod, P. Jacquemin, S. Atalick, and R. Herring, “An algorithm for 3-D refractive index measurement in holographic confocal microscopy,” Ultramicroscopy 107(2-3), 196–201 (2007).
[Crossref] [PubMed]

Other (3)

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

D. J. Whitehouse, Handbook of Surface and Nanometrology (CRC Press, 2011).

W. Osten, Optical Inspection of Microsystems (CRC Press, 2007).

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Figures (4)

Fig. 1
Fig. 1 Implementation of SOH in a confocal microscope. (a) Setup of a SOH confocal microscope. Laser: consisting of a stabilized HeNe laser, Faraday isolator and beam expander, BS: beam-splitter (50:50 non-polarizing), L1: microscope objective (20x, 0.4 NA Nikon E Plan), L2: Lens (f = 25.4mm), PH: pin hole (100 μm diameter for the experiments in Figs. 2 and 3 and 200 μm in case of Fig. 4), PZM: Piezo-actuated mirror, DET: one-pixel photodetector. (b) Linear movement of the reference mirror PZM synthesizes a reference wave UR(x,y) analogous to the plane reference wave in off-axis wide-field holography. Top: Simulated map of the mirror position d(x,y) consisting of 64 x 64 pixels, where we assumed vx = 1 mm/s, X = 1 mm, vR = 1/16 λ/s, Δy = 1/64 mm. Bottom: Resulting phase map φR(x,y) of the synthesized plane reference wave with kx = 2π/8 mm−1 and ky = 16∙2π mm−1.
Fig. 2
Fig. 2 Demonstration of SOH confocal microscopy with a test sample. (a) Schematic cross section of the test sample exhibiting pits of various depths (10 μm x 8 μm area, depth is indicated by the numbers below). (b) Non-interferometric confocal image. (c) Confocal synthetic hologram I(r) , (798 x 195 pixel, 80 μm x 20 μm, imaging time 195 sec, normalized to the intensity on the gold surface outside the holes). (d) Magnitude of Fourier-transform, | I ˜ (q)| , of I(r) (logarithmic color scale). The red dashed line shows the width of the window function applied in the reconstruction process. (e) Reconstructed amplitude image A S (r) . (f) Reconstructed phase image φ S (r) . (g) Corrected phase image Φ S (r) . (h) Zoom into the region outlined by the dashed square in (c). (i) Zoom of center part of | I ˜ (q)| showing the conjugate term A R U ˜ S * , autocorrelation term C and the direct term A R * U ˜ S (from top to bottom). (j,k) Zooms into the regions outlined by the dashed squares in (e,g), showing the amplitude and corrected phase, A S (r) and Φ S (r) .
Fig. 3
Fig. 3 Optically-obtained surface profile of the test sample. (a,b) Surface profiles obtained with SOH confocal microscopy, showings pits #1 to #4 and #5 to #8 (c,d) AFM topography images of pits #1 to #4 and #5 to #8. (e,f) Line profiles taken across the pits as indicated by the white arrows in (a-d). The optical line profiles (marked by ‘SOH’, red lines) represent data from a single line in (a,b), the AFM line profiles (black lines) represent the average over 10 neighboring lines in (c,d). (g) Zooms of the line profiles of pits #1 and #5 from (e) and (f), respectively. (h) Zoom of the optically obtained surface profile of pit #8 from (b). (i) Zoom of the AFM topography of pit #8 from (d). (j) Line profiles taken from (h,i) across pit #8 as indicated by the white arrows. The black arrow in (h) points to a region exhibiting thickness variations of the Au cladding that are resolved in the optically-obtained surface profile as well as in AFM topography (see text).
Fig. 4
Fig. 4 Surface profiling of an onion cell. (a) Schematic. (b) Conventional, non-inteferometric confocal image of the surface of a single onion cell. The cell is aligned in y-direction and only part of the cell is shown with the cell walls visible at the left and right border. (c-d) Confocal amplitude and phase images, A S (r) and φ S (r) , obtained with SOH (1500 x 1500 pixel, 100 μm x 100 μm, imaging time 25 min). (e) Surface profile of the onion cell h(r) . (f) High-pass filtered surface profile H(r) to reveal details on the surface. (g,h) Line profiles taken along the dashed line in (e) and (f), respectively.

Equations (4)

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I(r)= | U R (r) | 2 + | U S (r) | 2 + U S * (r) U R (r)+ U R * (r) U S (r).
k x = 4π λ v R v x and k y = 4π λ v R Δ y v x /2X ,
I ˜ (q)=C(q)+ A R U ˜ S * ( k || q )+ A R * U ˜ S ( k || +q ),
h(r)= λ 22π Φ S (r),

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