Abstract

A strategy is presented to enable optical-sectioning microscopy with improved contrast and imaging depth using low-power (0.5 - 1 mW) diode laser illumination. This technology combines the inherent strengths of focal-modulation microscopy and dual-axis confocal (DAC) microscopy for rejecting out-of-focus and multiply scattered background light in tissues. The DAC architecture is unique in that it utilizes an intersecting pair of illumination and collection beams to improve the spatial-filtering and optical-sectioning performance of confocal microscopy while focal modulation selectively ‘labels’ in-focus signals via amplitude modulation. Simulations indicate that modulating the spatial alignment of dual-axis beams at a frequency f generates signals from the focal volume of the microscope that are modulated at 2f with minimal modulation of background signals, thus providing nearly an order-of-magnitude improvement in optical-sectioning contrast compared to DAC microscopy alone. Experiments show that 2f lock-in detection enhances contrast and imaging depth within scattering phantoms and fresh tissues.

© 2014 Optical Society of America

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2013 (2)

2012 (6)

2011 (1)

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

2010 (2)

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

2008 (2)

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

N. Chen, C. H. Wong, and C. J. R. Sheppard, “Focal modulation microscopy,” Opt. Express16(23), 18764–18769 (2008).
[CrossRef] [PubMed]

2007 (2)

L. K. Wong, M. J. Mandella, G. S. Kino, and T. D. Wang, “Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture,” Opt. Lett.32(12), 1674–1676 (2007).
[CrossRef] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

2006 (3)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

A. Bilenca, A. Ozcan, B. Bouma, and G. Tearney, “Fluorescence coherence tomography,” Opt. Express14(16), 7134–7143 (2006).
[CrossRef] [PubMed]

2005 (1)

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2004 (1)

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

2003 (2)

O. De Wever and M. Mareel, “Role of tissue stroma in cancer cell invasion,” J. Pathol.200(4), 429–447 (2003).
[CrossRef] [PubMed]

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

2001 (2)

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

1997 (1)

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

1994 (1)

E. H. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun.111(5-6), 536–547 (1994).
[CrossRef]

1991 (1)

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1941 (1)

L. G. Henyey and J. L. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J.93, 70–83 (1941).
[CrossRef]

Ballini, J. P.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Bates, M.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Berning, S.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

Betzig, E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bilenca, A.

Boas, D. A.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bouma, B.

Braichotte, D.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Brooks, D. H.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Chang, W.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Chen, N.

Chen, Y.

Cheng, S.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Contag, C. H.

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Crawford, J. M.

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

De Wever, O.

O. De Wever and M. Mareel, “Role of tissue stroma in cancer cell invasion,” J. Pathol.200(4), 429–447 (2003).
[CrossRef] [PubMed]

Del Bene, F.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Dempsey, G. T.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dibaj, P.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

DiMarzio, C. A.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Dwyer, P. J.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

et,

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Fahrbach, F. O.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun.3, 632 (2012).
[CrossRef] [PubMed]

Flotte, T.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Friedland, S.

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Gaudette, R. J.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

González, S.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Greenstein, J. L.

L. G. Henyey and J. L. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J.93, 70–83 (1941).
[CrossRef]

Gregory, K.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Haeberle, H.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

Hee, M. R.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Hell, S. W.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

Henyey, L. G.

L. G. Henyey and J. L. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J.93, 70–83 (1941).
[CrossRef]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Huang, B.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Huang, D.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Huisken, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Isobe, K.

Ji, N.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

Kawano, H.

Kilmer, M.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Kino, G. S.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

L. K. Wong, M. J. Mandella, G. S. Kino, and T. D. Wang, “Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture,” Opt. Lett.32(12), 1674–1676 (2007).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Ku, G.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Kumagai, A.

Leigh, S. Y.

Levenson, R. M.

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

Lin, C. P.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Lindek, S.

E. H. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun.111(5-6), 536–547 (1994).
[CrossRef]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Liu, J. T. C.

D. Wang, Y. Chen, Y. Wang, and J. T. C. Liu, “Comparison of line-scanned and point-scanned dual-axis confocal microscope performance,” Opt. Lett.38(24), 5280–5283 (2013).
[CrossRef] [PubMed]

D. Wang, Y. Chen, and J. T. C. Liu, “A liquid optical phantom with tissue-like heterogeneities for confocal microscopy,” Biomed. Opt. Express3(12), 3153–3160 (2012).
[CrossRef] [PubMed]

S. Y. Leigh and J. T. C. Liu, “Multi-color miniature dual-axis confocal microscope for point-of-care pathology,” Opt. Lett.37(12), 2430–2432 (2012).
[CrossRef] [PubMed]

Y. Chen, D. Wang, and J. T. C. Liu, “Assessing the tissue-imaging performance of confocal microscope architectures via Monte Carlo simulations,” Opt. Lett.37(21), 4495–4497 (2012).
[CrossRef] [PubMed]

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Loewke, N. O.

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

Mandella, M. J.

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

L. K. Wong, M. J. Mandella, G. S. Kino, and T. D. Wang, “Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture,” Opt. Lett.32(12), 1674–1676 (2007).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Mareel, M.

O. De Wever and M. Mareel, “Role of tissue stroma in cancer cell invasion,” J. Pathol.200(4), 429–447 (2003).
[CrossRef] [PubMed]

Menaker, G.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Midorikawa, K.

Milkie, D. E.

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

Miller, E. L.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Miyawaki, A.

Mizuno, H.

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Ozcan, A.

Pang, Y.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Piyawattanametha, W.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

Puliafito, C. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Ra, H.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

Rajadhyaksha, M.

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

Rohrbach, A.

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun.3, 632 (2012).
[CrossRef] [PubMed]

Schuman, J. S.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Sheppard, C. J. R.

Soetikno, R.

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Solgaard, O.

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Steffens, H.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

Stelzer, E. H.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

E. H. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun.111(5-6), 536–547 (1994).
[CrossRef]

Stinson, W. G.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Stoica, G.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Suda, A.

Swanson, E. A.

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

Swoger, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Takeda, T.

Tearney, G.

Utke, N.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

van den Bergh, H.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Wagnières, G.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Wang, D.

Wang, L. V.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Wang, T. D.

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

L. K. Wong, M. J. Mandella, G. S. Kino, and T. D. Wang, “Improved rejection of multiply scattered photons in confocal microscopy using dual-axes architecture,” Opt. Lett.32(12), 1674–1676 (2007).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

Wang, X.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Wang, Y.

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Willig, K. I.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

Wittbrodt, J.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

Wong, C. H.

Wong, L. K.

Xie, X.

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Zellweger, M.

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

Zhang, Q.

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

Zhuang, X.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

Anal. Cell Pathol. (Amst.) (1)

J. T. C. Liu, N. O. Loewke, M. J. Mandella, R. M. Levenson, J. M. Crawford, and C. H. Contag, “Point-of-care pathology with miniature microscopes,” Anal. Cell Pathol. (Amst.)34(3), 81–98 (2011).
[PubMed]

Astrophys. J. (1)

L. G. Henyey and J. L. Greenstein, “Diffuse radiation in the galaxy,” Astrophys. J.93, 70–83 (1941).
[CrossRef]

Biomed. Opt. Express (3)

IEEE Sig. Proc. Mag. (1)

D. A. Boas, D. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J. Gaudette, and Q. Zhang, “Imaging the body with diffuse optical tomography,” IEEE Sig. Proc. Mag.18(6), 57–75 (2001).
[CrossRef]

J. Biomed. Opt. (3)

J. T. C. Liu, M. J. Mandella, J. M. Crawford, C. H. Contag, T. D. Wang, and G. S. Kino, “Efficient rejection of scattered light enables deep optical sectioning in turbid media with low-numerical-aperture optics in a dual-axis confocal architecture,” J. Biomed. Opt.13(3), 034020 (2008).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, S. Friedland, R. Soetikno, J. M. Crawford, C. H. Contag, G. S. Kino, and T. D. Wang, “Dual-axes confocal reflectance microscope for distinguishing colonic neoplasia,” J. Biomed. Opt.11(5), 054019 (2006).
[CrossRef] [PubMed]

J. T. C. Liu, M. J. Mandella, N. O. Loewke, H. Haeberle, H. Ra, W. Piyawattanametha, O. Solgaard, G. S. Kino, and C. H. Contag, “Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery,” J. Biomed. Opt.15(2), 026029 (2010).
[CrossRef] [PubMed]

J. Invest. Dermatol. (1)

M. Rajadhyaksha, G. Menaker, T. Flotte, P. J. Dwyer, and S. González, “Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide Mohs micrographic surgery without frozen histopathology,” J. Invest. Dermatol.117(5), 1137–1143 (2001).
[CrossRef] [PubMed]

J. Pathol. (1)

O. De Wever and M. Mareel, “Role of tissue stroma in cancer cell invasion,” J. Pathol.200(4), 429–447 (2003).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

X. Wang, Y. Pang, G. Ku, X. Xie, G. Stoica, and L. V. Wang, “Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain,” Nat. Biotechnol.21(7), 803–806 (2003).
[CrossRef] [PubMed]

Nat. Commun. (1)

F. O. Fahrbach and A. Rohrbach, “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat. Commun.3, 632 (2012).
[CrossRef] [PubMed]

Nat. Methods (1)

N. Ji, D. E. Milkie, and E. Betzig, “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nat. Methods7(2), 141–147 (2010).
[CrossRef] [PubMed]

Opt. Commun. (1)

E. H. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun.111(5-6), 536–547 (1994).
[CrossRef]

Opt. Express (2)

Opt. Lett. (4)

Phys. Med. Biol. (1)

G. Wagnières, S. Cheng, M. Zellweger, N. Utke, D. Braichotte, J. P. Ballini, and H. van den Bergh, “An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy,” Phys. Med. Biol.42(7), 1415–1426 (1997).
[CrossRef] [PubMed]

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

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Science (6)

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor super-resolution imaging with photo-switchable fluorescent probes,” Science317(5845), 1749–1753 (2007).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a living mouse brain,” Science335(6068), 551 (2012).
[CrossRef] [PubMed]

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science305(5686), 1007–1009 (2004).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990).
[CrossRef] [PubMed]

D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et, “Optical coherence tomography,” Science254(5035), 1178–1181 (1991).
[CrossRef] [PubMed]

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Figures (8)

Fig. 1
Fig. 1

Resolution and depth tradeoffs in common optical imaging modalities. In general, deeper imaging requires sacrificing spatial resolution. Approximate lower and upper bounds on these parameters are mapped for single-axis confocal microscopy (SAC), selective plane illumination microscopy (SPIM), optical coherence tomography (OCT), two-photon microscopy (2P), dual-axis confocal microscopy (DAC), photoacoustic tomography (PAT) and diffuse optical tomography (DOT). Note that modulated-alignment dual-axis (MAD) confocal microscopy seeks to extend DAC’s penetration depth at minimal cost to resolution. Depth of imaging is expressed in terms of mean free path (MFP = 1/μs) and transport mean free path (TMFP = 1/μs’), where μs’ = μs(1 – g). Since the anisotropy factor of tissue, g, is typically ~0.9, the transport mean free path is typically one order of magnitude larger than the mean free path (TMFP ~10MFP).

Fig. 2
Fig. 2

Principles of modulated-alignment dual-axis (MAD) confocal microscopy. By periodically sweeping the illumination beam into (a) and out of alignment (b) with the collection beam at a given frequency f, the signal generated at the focal volume experiences an amplitude modulation at 2f (c) and can therefore be distinguished from background signal that remains largely unmodulated.

Fig. 3
Fig. 3

MAD architecture and optical circuit. (a) The MAD system is built by modifying a typical dual-axis confocal (DAC) tabletop microscope. An acousto-optical deflector (AOD), which is optimized to produce a 1st-order diffracted beam, is inserted into the optical path upstream of the illumination arm. The 1st diffracted order serves as the illumination beam in the MAD system. A slit (S1) oriented perpendicularly to the dispersion direction is used to isolate the 1st-order beam and to block all other diffracted orders. Pixel intensities correspond to the magnitude of the 2f signal. (b) A photograph of the tabletop MAD setup. (c) Motion of the illumination beam is minimized in relation to the motion of the focus (right side of ray-trace diagram) by ensuring that the pivot point about which the illumination beam rotates (for alignment modulation) is located near or at the focusing objective. This ensures that the motion of the illumination beam at the focus is larger than the motion of the beam prior to the focus.

Fig. 4
Fig. 4

Optimizing modulation depth. (a) In response to a mirror placed at the focal plane, the optical throughput of the microscope system decays as the alignment offset between the illumination and detection paths increases. (b) When sinusoidally modulating the alignment offset at a modulation depth of amplitude Δy/ω0, the maximum 2f signal is generated at Δy/ω0 ~1.5 - 1.8. Example time traces (c) and their frequency content (d) at three distinct modulation depths demonstrate the dependency of the 2f signal on modulation depth.

Fig. 5
Fig. 5

Monte-Carlo simulations. (a) Typical DAC microscope system geometry where θ is the crossing half-angle between the beams and α is the focusing half-angle of each beam. (b) A ~9-dB improvement in optical-sectioning contrast is achieved at all depths (perpendicular optical lengths. Lp, ranging from 2 to 10). (c) Monte-Carlo simulations show that the optimal modulation depth for maximizing contrast (signal to background ratio, SBR) is consistently Δy/ω0 ~1.5 - 1.8 over a range of imaging depths (or perpendicular optical lengths, Lp).

Fig. 6
Fig. 6

Axial and transverse responses of MAD and DAC in scattering media. Intralipid experiments demonstrate consistent improvement in optical sectioning contrast in the MAD technique over DAC. (a) Contrast improves by 5 - 6 dB axially without compromising optical section thickness. (b) In-plane resolution is maintained while improving contrast by up to ~4 dB. The signal labeled as 'Water' refers to characterization of the system in the absence of scattering (whereas scattering is achieved using Intralipid).

Fig. 7
Fig. 7

MAD imaging performance in fluorescent phantoms. Vertical and en face images acquired at a depth of ~200 μm demonstrate the MAD technique’s ability to reduce the amount of background fluorescence due to out-of-focus and multiply scattered light, thereby improving the contrast when visualizing bright fluorescent beads in a dim fluorescent matrix. Line profiles (bottom left) in the y-direction through selected fluorescent beads from the en face images show improved contrast. Scale bars measure 20 μm.

Fig. 8
Fig. 8

MAD imaging performance in fresh tissues. En face images from fresh mouse tissues demonstrate distinct contrast enhancement in MAD compared with DAC (white arrows indicate example features). Imaging depths for the intestine, renal capsule and renal medulla examples are 25 μm, 80 μm, and 60 μm, respectively. Scale bars measure 40 μm.

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