Abstract

It is well known that the principle of reciprocity is valid for light traveling even through scattering or absorptive media. This principle has been used to establish an equivalence between conventional widefield microscopes and scanning microscopes. We make use of this principle to introduce a scanning version of oblique back-illumination microscopy, or sOBM. This technique provides sub-surface phase-gradient and amplitude images from unlabeled tissue, in an epi-detection geometry. That is, it may be applied to arbitrarily thick tissue. sOBM may be implemented as a simple, cost-effective add-on with any scanning microscope, requiring only the availability of an extra input channel in the microscope electronics. We demonstrate here its implementation in combination with two-photon excited fluorescence (TPEF) microscopy and with coherent anti-Stokes Raman scattering (CARS) microscopy, applied to brain or spinal cord tissue imaging. In both cases, sOBM provides information on tissue morphology complementary to TPEF or CARS contrast. This information is obtained simultaneously and is automatically co-registered. Finally, we show that sOBM can be operated at video rate.

© 2014 Optical Society of America

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2013 (1)

T. N. Ford and J. Mertz, “Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy,” J. Biomed Opt.18, 0066007 (2013).
[CrossRef]

2012 (2)

2011 (1)

2009 (1)

2008 (2)

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

2006 (2)

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

R. Yi, K. K. Chu, and J. Mertz, “Graded-field microscopy with white light,” Opt. Express14, 5191–5200 (2006).
[CrossRef] [PubMed]

2005 (1)

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

2004 (1)

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

2003 (1)

J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotech.21, 1361–1367 (2003).
[CrossRef]

1999 (2)

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett.82, 4142–4145 (1999).
[CrossRef]

H. U. Dodt, M. Eder, A. Frick, and W. Zieglg¨ansberger, “Precisely localized ltd in the neocortex revealed by infrared-guided laser stimulation,” Science286, 110–113 (1999).
[CrossRef] [PubMed]

1990 (1)

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

1986 (1)

C. J. R. Sheppard and T. Wilson, “On the equivalence of scanning and conventional microscopes,” Optik73, 39–43 (1986).

1985 (1)

B. Kachar, “Asymmetric illumination contrast: a method of image formation for video microscopy,” Science227, 766–768 (1985).
[CrossRef] [PubMed]

1977 (2)

1975 (1)

1973 (1)

M. E. Barnett, “The reciprocity theorem and the equivalence of conventional and transmission microscopes,” Optik38, 585–588 (1973).

1972 (1)

W. T. Welford, “On the relationship between the modes of image formation in scanning microscopy and conventional microscopy,” J. Microsc.96, 105–107 (1972).
[CrossRef] [PubMed]

1955 (2)

F. Zernike, “How I discovered phase contrast,” Science121, 345–349 (1955).
[CrossRef] [PubMed]

G. Nomarski, “Microinterferomètre différentielà ondes polarisées,” J. Phys. Radium16, S9 (1955).

1873 (1)

Lord Rayleigh, “Some general theorems relating to vibrations,” Proc. Lond. Math. Soc.4, 357–368 (1873).

Barnett, M. E.

M. E. Barnett, “The reciprocity theorem and the equivalence of conventional and transmission microscopes,” Optik38, 585–588 (1973).

Bélanger, E.

Biss, D. P.

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

Brecht, M.

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Burns, S.

Chu, K. K.

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Meth.9, 1195–1197 (2012).
[CrossRef]

R. Yi, K. K. Chu, and J. Mertz, “Graded-field microscopy with white light,” Opt. Express14, 5191–5200 (2006).
[CrossRef] [PubMed]

Chui, T. Y. P.

Coté, D.

E. Bélanger, F. P. Henry, R. Vallée, M. A. Randolph, I. E. Kochevar, J. M. Winograd, C. P. Lin, and D. Coté, “In vivo evaluation of demyelination and remyelination in a nerve crush injury model,” Biomed. Opt. Express2, 2698–2708 (2011).
[CrossRef] [PubMed]

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

Denk, W.

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

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

Dodd, J.

Dodt, H. U.

H. U. Dodt, M. Eder, A. Frick, and W. Zieglg¨ansberger, “Precisely localized ltd in the neocortex revealed by infrared-guided laser stimulation,” Science286, 110–113 (1999).
[CrossRef] [PubMed]

Eder, M.

H. U. Dodt, M. Eder, A. Frick, and W. Zieglg¨ansberger, “Precisely localized ltd in the neocortex revealed by infrared-guided laser stimulation,” Science286, 110–113 (1999).
[CrossRef] [PubMed]

Evans, C. L.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

Fee, M. S.

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Ford, T. N.

T. N. Ford and J. Mertz, “Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy,” J. Biomed Opt.18, 0066007 (2013).
[CrossRef]

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Meth.9, 1195–1197 (2012).
[CrossRef]

Frick, A.

H. U. Dodt, M. Eder, A. Frick, and W. Zieglg¨ansberger, “Precisely localized ltd in the neocortex revealed by infrared-guided laser stimulation,” Science286, 110–113 (1999).
[CrossRef] [PubMed]

Fujimoto, J. G.

J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotech.21, 1361–1367 (2003).
[CrossRef]

Garaschuk, O.

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Gross, L.

Ha, M.

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

Helmchen, F.

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

F. Helmchen, A. Konnerth, and R. Yuste, Imaging in Neuroscience: A Laboratory Manual, 2nd ed. (CSHL Press, 2011).

Henry, F. P.

Hoffman, R.

Holtom, G. R.

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett.82, 4142–4145 (1999).
[CrossRef]

Judkewitz, B.

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

Kachar, B.

B. Kachar, “Asymmetric illumination contrast: a method of image formation for video microscopy,” Science227, 766–768 (1985).
[CrossRef] [PubMed]

Kano, M.

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

Kermisch, D.

Kitamura, K.

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

Kochevar, I. E.

Komai, S.

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

Konnerth, A.

F. Helmchen, A. Konnerth, and R. Yuste, Imaging in Neuroscience: A Laboratory Manual, 2nd ed. (CSHL Press, 2011).

Lin, C. P.

E. Bélanger, F. P. Henry, R. Vallée, M. A. Randolph, I. E. Kochevar, J. M. Winograd, C. P. Lin, and D. Coté, “In vivo evaluation of demyelination and remyelination in a nerve crush injury model,” Biomed. Opt. Express2, 2698–2708 (2011).
[CrossRef] [PubMed]

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

Margrie, T. W.

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Masters, B. R.

B. R. Masters and P. So, Handbook of Biomedical Nonlinear Optical Microscopy, 1st ed. (Oxford Univ. Press, 2008).

Mehta, S. B.

Mertz, J.

T. N. Ford and J. Mertz, “Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy,” J. Biomed Opt.18, 0066007 (2013).
[CrossRef]

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Meth.9, 1195–1197 (2012).
[CrossRef]

R. Yi, K. K. Chu, and J. Mertz, “Graded-field microscopy with white light,” Opt. Express14, 5191–5200 (2006).
[CrossRef] [PubMed]

Nomarski, G.

G. Nomarski, “Microinterferomètre différentielà ondes polarisées,” J. Phys. Radium16, S9 (1955).

Osten, P.

S. Komai, W. Denk, P. Osten, M. Brecht, and T. W. Margrie, “Two-photon targeted patching (tptp) in vivo,” Nat. Protoc.1, 647–652 (2006).
[CrossRef]

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Pawley, J.

J. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, 2006).
[CrossRef]

Potma, E. O.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

Puoris’haag, M.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

Randolph, M. A.

Rayleigh, Lord

Lord Rayleigh, “Some general theorems relating to vibrations,” Proc. Lond. Math. Soc.4, 357–368 (1873).

Sheppard, C. J. R.

So, P.

B. R. Masters and P. So, Handbook of Biomedical Nonlinear Optical Microscopy, 1st ed. (Oxford Univ. Press, 2008).

Spenser, J. A.

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

Strickler, J. H.

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

Svoboda, K.

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

Tuchin, V.

V. Tuchin, Tissue Optics: Light scattering methods and instruments for medical diagnosis, 2nd ed. (SPIE Publications, 2007).
[CrossRef]

Vallée, R.

VanNasdale, D. A.

Veilleux, I.

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

von Helmholtz, H.

H. von Helmholtz, Handbuch der physiologischen Optik,1st ed. (Leopold Voss, Leipzig, 1856).

Webb, W. W.

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

Welford, W. T.

W. T. Welford, “On the relationship between the modes of image formation in scanning microscopy and conventional microscopy,” J. Microsc.96, 105–107 (1972).
[CrossRef] [PubMed]

Wilson, T.

C. J. R. Sheppard and T. Wilson, “On the equivalence of scanning and conventional microscopes,” Optik73, 39–43 (1986).

Winograd, J. M.

Xie, X. S.

C. L. Evans, E. O. Potma, M. Puoris’haag, D. Coté, C. P. Lin, and X. S. Xie, “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc. Natl. Acad. Sci. USA102, 16807–16812 (2005).
[CrossRef] [PubMed]

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett.82, 4142–4145 (1999).
[CrossRef]

Yi, R.

Yuste, R.

F. Helmchen, A. Konnerth, and R. Yuste, Imaging in Neuroscience: A Laboratory Manual, 2nd ed. (CSHL Press, 2011).

Zernike, F.

F. Zernike, “How I discovered phase contrast,” Science121, 345–349 (1955).
[CrossRef] [PubMed]

Zieglg¨ansberger, W.

H. U. Dodt, M. Eder, A. Frick, and W. Zieglg¨ansberger, “Precisely localized ltd in the neocortex revealed by infrared-guided laser stimulation,” Science286, 110–113 (1999).
[CrossRef] [PubMed]

Zumbusch, A.

A. Zumbusch, G. R. Holtom, and X. S. Xie, “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett.82, 4142–4145 (1999).
[CrossRef]

Appl. Opt. (2)

Biomed. Opt. Express (2)

IEEE J. Sel. Top. Quantum Electron. (1)

I. Veilleux, J. A. Spenser, D. P. Biss, D. Coté, and C. P. Lin, “In vivo cell tracking with video rate multimodality laser scanning microscopy,” IEEE J. Sel. Top. Quantum Electron.14, 10–18 (2008).
[CrossRef]

J. Microsc. (1)

W. T. Welford, “On the relationship between the modes of image formation in scanning microscopy and conventional microscopy,” J. Microsc.96, 105–107 (1972).
[CrossRef] [PubMed]

J. Biomed Opt. (1)

T. N. Ford and J. Mertz, “Video-rate imaging of microcirculation with single-exposure oblique back-illumination microscopy,” J. Biomed Opt.18, 0066007 (2013).
[CrossRef]

J. Neurosci. (1)

M. Brecht, M. S. Fee, O. Garaschuk, F. Helmchen, T. W. Margrie, K. Svoboda, and P. Osten, “Novel approaches to monitor and manipulate single neurons in vivo,” J. Neurosci.24, 9223–9228 (2004).
[CrossRef] [PubMed]

J. Opt. Soc. Am. (1)

J. Phys. Radium (1)

G. Nomarski, “Microinterferomètre différentielà ondes polarisées,” J. Phys. Radium16, S9 (1955).

Nat. Biotech. (1)

J. G. Fujimoto, “Optical coherence tomography for ultrahigh resolution in vivo imaging,” Nat. Biotech.21, 1361–1367 (2003).
[CrossRef]

Nat. Meth. (2)

T. N. Ford, K. K. Chu, and J. Mertz, “Phase-gradient microscopy in thick tissue with oblique back-illumination,” Nat. Meth.9, 1195–1197 (2012).
[CrossRef]

K. Kitamura, B. Judkewitz, M. Kano, W. Denk, and M. Ha, “Targeted patch-clamp recordings and single-cell electroporation of unlabeled neurons in vivo,” Nat. Meth.5, 61–67 (2008).
[CrossRef]

Nat. Protoc. (1)

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Supplementary Material (1)

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Figures (4)

Fig. 1
Fig. 1

Schematics of OBM and sOBM setups. In the case of OBM, illumination is delivered into a scattering sample via off axis optical fibers. Oblique backscattered illumination traverses the focal plane and is collected by the microscope objective, whereupon it is ultimately detected by a camera. In the case of sOBM, a scanning laser beam is focused into a scattering sample. The illumination traverses the focal plane and is backscattered, whereupon it is collected by off axis optical fibers and detected by photodiodes. In both cases, the fibers are held flush with the sample surface by a support ring (not shown).

Fig. 2
Fig. 2

Simultaneous TPEF (a) and sOBM (b) images of a brain slice of mouse cortex where specific classes of excitatory neurons are genetically labeled with GFP, and overlay (c), taken from a z stack ( Media 1 – step size 2 μm). Note non-fluorescent structures revealed by sOBM include unlabeled neurons, dendritic processes receiving synaptic contacts, and a blood vessel. Horizontal processes can be revealed by sOBM but vertical cannot, because of the orientation of the fibers. Focal depth is approximately 30 μm. Field of view is approximately 80 μm. Panel (d) is the z-stack of a representative single fluorescent cell (top right in panel (a)), acquired by TPEF (above) and sOBM (below). Note similar lateral and axial resolutions.

Fig. 3
Fig. 3

Simultaneous CARS (left) and sOBM (right) images of a cross-sectional cut of a mouse spinal cord from lumbar region. The CARS signal arose mostly from lipid-rich myelin sheaths surrounding nerve axons. Note correspondence between CARS and sOBM images. Focal depth was approximately 10 μm. Field of view was (a–d) 78 μm and (e,f) 55 μm.

Fig. 4
Fig. 4

Images of a coronal section of mouse brain acquired by video-rate sOBM. Panel (a) reveals cell bodies from gray matter; panel (b) reveals axon tracts likely to be myelinated. Focal depth was approximately 10μm. Field of view was approximately 254 × 159 μm.

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