Abstract

Confocal laser microscopy (CLM) is a powerful tool in life science research and industrial inspection because it offers two-dimensional optical sectioning or three-dimensional imaging capability with micrometer depth selectivity. Furthermore, scan-less imaging modality enables rapid image acquisition and high robustness against surrounding external disturbances in CLM. However, the objects to be measured must be reflective, absorptive, scattering, or fluorescent because the image contrast is given by the optical intensity. If a new image contrast can be provided by the optical phase, scan-less CLM can be further applied for transparent non-fluorescent objects or reflective objects with nanometer unevenness by providing information on refractive index, optical thickness, or geometrical shape. Here, we report scan-less confocal dual-comb microscopy offering a phase image in addition to an amplitude image with depth selectivity by using an optical frequency comb as an optical carrier of amplitude and phase with discrete ultra-multichannels. Our technique encodes confocal amplitude and phase images of a sample onto a series of discrete modes in the optical frequency comb with well-defined amplitude and phase to establish a one-to-one correspondence between image pixels and comb modes. The technique then decodes these images from comb modes with amplitude and phase. We demonstrate confocal phase imaging with milliradian phase resolution under micrometer depth selectivity on the millisecond timescale. As a proof of concept, we demonstrate the quantitative phase imaging of standing culture fixed cells and the surface topography of nanometer-scale step structures. Our technique for confocal phase imaging will find applications in three-dimensional visualization of stacked living cells in culture and nanometer surface topography of semiconductor objects.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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References

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2017 (1)

2016 (7)

2015 (1)

2014 (2)

T. Ideguchi, A. Poisson, G. Guelachvili, N. Picqué, and T. W. Hänsch, “Adaptive real-time dual-comb spectroscopy,” Nat. Commun. 5, 3375 (2014).
[Crossref]

Y.-D. Hsieh, Y. Iyonaga, Y. Sakaguchi, S. Yokoyama, H. Inaba, K. Minoshima, F. Hindle, T. Araki, and T. Yasui, “Spectrally interleaved, comb-mode-resolved spectroscopy using swept dual terahertz combs,” Sci. Rep. 4, 3816 (2014).
[Crossref]

2012 (2)

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

J. Roy, J.-D. Deschênes, S. Potvin, and J. Genest, “Continuous real-time correction and averaging for frequency comb interferometry,” Opt. Express 20, 21932–21939 (2012).
[Crossref]

2011 (1)

E. Baumann, F. R. Giorgetta, W. C. Swann, A. M. Zolot, I. Coddington, and N. R. Newbury, “Spectroscopy of the methane v3 band with an accurate midinfrared coherent dual-comb spectrometer,” Phys. Rev. A 84, 062513 (2011).
[Crossref]

2010 (2)

2009 (2)

K. K. Tsia, K. Goda, D. Capewell, and B. Jalali, “Simultaneous mechanical-scan-free confocal microscopy and laser microsurgery,” Opt. Lett. 34, 2099–2101 (2009).
[Crossref]

K. Goda, K. K. Tsia, and B. Jalali, “Serial time-encoded amplified imaging for real-time observation of fast dynamic phenomena,” Nature 458, 1145–1149 (2009).
[Crossref]

2007 (2)

M. T. Tilli, M. C. Cabrera, A. R. Parrish, K. M. Torre, M. K. Sidawy, A. L. Gallagher, E. Makariou, S. A. Polin, M. C. Liu, and P. A. Furth, “Real-time imaging and characterization of human breast tissue by reflectance confocal microscopy,” J. Biomed. Opt. 12, 051901 (2007).
[Crossref]

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445, 627–630 (2007).
[Crossref]

2006 (2)

T. Yasui, Y. Kabetani, E. Saneyoshi, S. Yokoyama, and T. Araki, “Terahertz frequency comb by multifrequency-heterodyning photoconductive detection for high-accuracy, high-resolution terahertz spectroscopy,” Appl. Phys. Lett. 88, 241104 (2006).
[Crossref]

G. Popescu, T. Ikeda, R. R. Dasari, and M. S. Feld, “Diffraction phase microscopy for quantifying cell structure and dynamics,” Opt. Lett. 31, 775–777 (2006).
[Crossref]

2005 (2)

C. J. Mann, L. Yu, C. M. Lo, and M. K. Kim, “High-resolution quantitative phase-contrast microscopy by digital holography,” Opt. Express 13, 8693–8698 (2005).
[Crossref]

A. Vogel, J. Noack, G. Hüttman, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B 81, 1015–1047 (2005).
[Crossref]

2004 (4)

2003 (1)

2002 (4)

G. J. Tearney, M. Shishkov, and B. E. Bouma, “Spectrally encoded miniature endoscopy,” Opt. Lett. 27, 412–414 (2002).
[Crossref]

S. Schiller, “Spectrometry with frequency combs,” Opt. Lett. 27, 766–768 (2002).
[Crossref]

D. Lellouchi, F. Beaudoin, C. Le Touze, P. Perdu, and R. Desplats, “IR confocal laser microscopy for MEMS technological evaluation,” Microelectron. Reliab. 42, 1815–1817 (2002).
[Crossref]

T. Udem, R. Holzwarth, and T. W. Hänsch, “Optical frequency metrology,” Nature 416, 233–237 (2002).
[Crossref]

2000 (1)

M. Niering, R. Holzwarth, J. Reichert, P. Pokasov, T. Udem, M. Weitz, T. W. Hänsch, P. Lemonde, G. Santarelli, M. Abgrall, P. Laurent, C. Salomon, and A. Clairon, “Measurement of the hydrogen 1S-2S transition frequency by phase coherent comparison with a microwave cesium fountain clock,” Phys. Rev. Lett. 84, 5496–5499 (2000).
[Crossref]

1999 (2)

T. Udem, J. Reichert, R. Holzwarth, and T. W. Hänsch, “Accurate measurement of large optical frequency differences with a mode-locked laser,” Opt. Lett. 24, 881–883 (1999).
[Crossref]

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol. 113, 293–303 (1999).
[Crossref]

1998 (1)

B. V. R. Tata and B. Raj, “Confocal laser scanning microscopy: applications in material science and technology,” Bull. Mater. Sci. 21(4), 263–278 (1998).
[Crossref]

1996 (1)

1994 (1)

K. Minoshima, H. Matsumoto, Z. Zhang, and T. Yagi, “Simultaneous 3-D imaging using chirped ultrashort optical pulses,” Jpn. J. Appl. Phys. 33, L1348–L1351 (1994).
[Crossref]

1984 (1)

R. L. Jungerman, P. C. D. Hobbs, and G. S. Kino, “Phase sensitive scanning optical microscope,” Appl. Phys. Lett. 45, 846–848 (1984).
[Crossref]

1979 (1)

G. J. Brakenhoff, P. Blom, and P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

1973 (1)

P. Davidovits and M. D. Egger, “Photomicrography of corneal endothelial cells in vivo,” Nature 244, 366–367 (1973).
[Crossref]

1942 (1)

F. Zernike, “Phase contrast, a new method for the microscopic observation of transparent objects,” Physica 9, 686–698 (1942).
[Crossref]

Abgrall, M.

M. Niering, R. Holzwarth, J. Reichert, P. Pokasov, T. Udem, M. Weitz, T. W. Hänsch, P. Lemonde, G. Santarelli, M. Abgrall, P. Laurent, C. Salomon, and A. Clairon, “Measurement of the hydrogen 1S-2S transition frequency by phase coherent comparison with a microwave cesium fountain clock,” Phys. Rev. Lett. 84, 5496–5499 (2000).
[Crossref]

Adam, J.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Anderson, R. R.

M. Rajadhyaksha, S. González, J. M. Zavislan, R. R. Anderson, and R. H. Webb, “In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology,” J. Invest. Dermatol. 113, 293–303 (1999).
[Crossref]

Araki, T.

Y.-D. Hsieh, Y. Iyonaga, Y. Sakaguchi, S. Yokoyama, H. Inaba, K. Minoshima, F. Hindle, T. Araki, and T. Yasui, “Spectrally interleaved, comb-mode-resolved spectroscopy using swept dual terahertz combs,” Sci. Rep. 4, 3816 (2014).
[Crossref]

T. Yasui, Y. Kabetani, E. Saneyoshi, S. Yokoyama, and T. Araki, “Terahertz frequency comb by multifrequency-heterodyning photoconductive detection for high-accuracy, high-resolution terahertz spectroscopy,” Appl. Phys. Lett. 88, 241104 (2006).
[Crossref]

Asahara, A.

Ayazi, A.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Badizadegan, K.

Barends, P.

G. J. Brakenhoff, P. Blom, and P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Baumann, E.

E. Baumann, F. R. Giorgetta, W. C. Swann, A. M. Zolot, I. Coddington, and N. R. Newbury, “Spectroscopy of the methane v3 band with an accurate midinfrared coherent dual-comb spectrometer,” Phys. Rev. A 84, 062513 (2011).
[Crossref]

Beaudoin, F.

D. Lellouchi, F. Beaudoin, C. Le Touze, P. Perdu, and R. Desplats, “IR confocal laser microscopy for MEMS technological evaluation,” Microelectron. Reliab. 42, 1815–1817 (2002).
[Crossref]

Blom, P.

G. J. Brakenhoff, P. Blom, and P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Bouma, B. E.

Brakenhoff, G. J.

G. J. Brakenhoff, P. Blom, and P. Barends, “Confocal scanning light microscopy with high aperture immersion lenses,” J. Microsc. 117, 219–232 (1979).
[Crossref]

Brown, R.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Cabrera, M. C.

M. T. Tilli, M. C. Cabrera, A. R. Parrish, K. M. Torre, M. K. Sidawy, A. L. Gallagher, E. Makariou, S. A. Polin, M. C. Liu, and P. A. Furth, “Real-time imaging and characterization of human breast tissue by reflectance confocal microscopy,” J. Biomed. Opt. 12, 051901 (2007).
[Crossref]

Capewell, D.

Chen, E.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Clairon, A.

M. Niering, R. Holzwarth, J. Reichert, P. Pokasov, T. Udem, M. Weitz, T. W. Hänsch, P. Lemonde, G. Santarelli, M. Abgrall, P. Laurent, C. Salomon, and A. Clairon, “Measurement of the hydrogen 1S-2S transition frequency by phase coherent comparison with a microwave cesium fountain clock,” Phys. Rev. Lett. 84, 5496–5499 (2000).
[Crossref]

Coddington, I.

I. Coddington, N. Newbury, and W. Swann, “Dual-comb spectroscopy,” Optica 3, 414–426 (2016).
[Crossref]

E. Baumann, F. R. Giorgetta, W. C. Swann, A. M. Zolot, I. Coddington, and N. R. Newbury, “Spectroscopy of the methane v3 band with an accurate midinfrared coherent dual-comb spectrometer,” Phys. Rev. A 84, 062513 (2011).
[Crossref]

Dasari, R. R.

Davidovits, P.

P. Davidovits and M. D. Egger, “Photomicrography of corneal endothelial cells in vivo,” Nature 244, 366–367 (1973).
[Crossref]

Deschênes, J.-D.

Desplats, R.

D. Lellouchi, F. Beaudoin, C. Le Touze, P. Perdu, and R. Desplats, “IR confocal laser microscopy for MEMS technological evaluation,” Microelectron. Reliab. 42, 1815–1817 (2002).
[Crossref]

Di Carlo, D.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Diddams, S. A.

S. A. Diddams, L. Hollberg, and V. Mbele, “Molecular fingerprinting with the resolved modes of a femtosecond laser frequency comb,” Nature 445, 627–630 (2007).
[Crossref]

Egger, M. D.

P. Davidovits and M. D. Egger, “Photomicrography of corneal endothelial cells in vivo,” Nature 244, 366–367 (1973).
[Crossref]

Fang-Yen, C.

Fard, A.

K. Goda, A. Mahjoubfar, C. Wang, A. Fard, J. Adam, D. R. Gossett, A. Ayazi, E. Sollier, O. Malik, E. Chen, Y. Liu, R. Brown, N. Sarkhosh, D. Di Carlo, and B. Jalali, “Hybrid dispersion laser scanner,” Sci. Rep. 2, 445 (2012).
[Crossref]

Feld, M. S.

Furth, P. A.

M. T. Tilli, M. C. Cabrera, A. R. Parrish, K. M. Torre, M. K. Sidawy, A. L. Gallagher, E. Makariou, S. A. Polin, M. C. Liu, and P. A. Furth, “Real-time imaging and characterization of human breast tissue by reflectance confocal microscopy,” J. Biomed. Opt. 12, 051901 (2007).
[Crossref]

Gallagher, A. L.

M. T. Tilli, M. C. Cabrera, A. R. Parrish, K. M. Torre, M. K. Sidawy, A. L. Gallagher, E. Makariou, S. A. Polin, M. C. Liu, and P. A. Furth, “Real-time imaging and characterization of human breast tissue by reflectance confocal microscopy,” J. Biomed. Opt. 12, 051901 (2007).
[Crossref]

Genest, J.

Gersen, H.

Giorgetta, F. R.

E. Baumann, F. R. Giorgetta, W. C. Swann, A. M. Zolot, I. Coddington, and N. R. Newbury, “Spectroscopy of the methane v3 band with an accurate midinfrared coherent dual-comb spectrometer,” Phys. Rev. A 84, 062513 (2011).
[Crossref]

Goda, K.

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Supplementary Material (5)

NameDescription
» Supplement 1       Supplemental Document
» Visualization 1       Propagation of a signal OFC, an image-encoded OFC, and a local OFC in DCM.
» Visualization 2       Kilohertz confocal amplitude and phase imaging obtained with a 1951 USAF resolution test chart moving in the x–y plane.
» Visualization 3       Series of confocal amplitude and phase images obtained with a 1951 USAF resolution test chart moving along the z direction.
» Visualization 4       Movie of 3D shape of the nanometer-scale three-step structure from various angles of vision.

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Figures (7)

Fig. 1.
Fig. 1. Principle of DCM. (a) Scan-less CLM based on 2D spectral encoding of OFC modes and (b) scan-less confocal amplitude and phase imaging by DCS.
Fig. 2.
Fig. 2. 2D spatial disperser. (a) Dependence of multiple transmission peaks on the y position in VIPA with a frequency spacing ΔνFSR and a frequency linewidth ΔνVIPA. (b) 2D spatial maps of OFC modes before and after passing through a diffraction grating. (c) 2D array of focal spots based on 2D spectral encoding of OFC modes.
Fig. 3.
Fig. 3. Experimental setup for DCM. See Section 2 for details. Visualization 1 illustrates the propagation of a signal OFC, an image-encoded OFC, and a local OFC in DCM.
Fig. 4.
Fig. 4. Mode-resolved amplitude and phase spectra of OFCs. (a) Schematic drawing of 1951 USAF resolution test chart with positive pattern. Mode-resolved (b) amplitude and (c) phase spectra of the no-image-encoded OFC. Mode-resolved (d) amplitude and (e) phase spectra of the image-encoded OFC. Normalized mode-resolved (f) amplitude and (g) phase spectra for spectral decoding of confocal amplitude and phase images, and their enlarged spectra of (h) amplitude and (i) phase.
Fig. 5.
Fig. 5. Confocal amplitude and phase images. (a) Confocal amplitude image and (b) confocal phase image of the test chart placed at the focal position (z=0  μm; image acquisition time, 81 ms). The relative phase was calculated by using a phase value at a certain pixel of the image as a reference. Visualization 2 shows a series of confocal amplitude and phase images of the test chart moving in the xy plane without signal accumulation (image acquisition time, 0.81 ms; image acquisition rate, 1234 Hz). (c) Transmission image of the test chart acquired by an infrared camera. (d) Confocal amplitude image and (e) confocal phase image of the test chart placed out of focus (z=+215  μm; image acquisition time, 81 ms). Visualization 3 shows the changes in the confocal amplitude and phase images when the sample position is moved along the z direction. (f) Confocal profile of the amplitude value with respect to the z position, which is calculated from the amplitude image in Visualization 3.
Fig. 6.
Fig. 6. Quantitative phase imaging of NIH3T3 cells. (a) Phase contrast image, (b) confocal amplitude image, and (c) confocal phase image of the sample placed at the focal position (z=0). The image acquisition time of the confocal amplitude and phase images was 81 ms.
Fig. 7.
Fig. 7. Surface topography based on confocal phase imaging. (a) Schematic drawing of a nanometer-scale three-step structure sample. (b) Confocal amplitude image and (c) confocal phase image of the sample placed at the focal position (z=0). The image acquisition time was 81 ms. (d) 3D image of the sample calculated from the confocal phase image. Visualization 4 shows the movie of the 3D shape from various angles of vision.

Equations (5)

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H(x,y)=12ϕ(x,y)2πλ=λ4πϕ(x,y),
Δz=0.88λnn2NA2=0.88×1.5501120.252=43  μm,
Δz=0.68λnn2NA2=0.68×1.5501.41.421.42=0.75  μm.
Δx=(fdθgdλ)ΔλFSR=11.4  μm,
Δy=(fdθVIPAdλ)ΔλVIPA=2.0  μm,

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