We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan.

© 2012 OSA

1. L. Sampath, S. Kwon, S. Ke, W. Wang, R. Schiff, M. E. Mawad, and E. M. Sevick-Muraca, “Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer,” J. Nucl. Med.48(9), 1501–1510 (2007). [CrossRef] [PubMed]
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Am. J. Physiol.

• I. J. LeGrice, B. H. Smaill, L. Z. Chai, S. G. Edgar, J. B. Gavin, and P. J. Hunter, “Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog,” Am. J. Physiol.269(2 Pt 2), H571–H582 (1995). [PubMed]

Ann. N. Y. Acad. Sci.

• S. L. K. Bowers, T. K. Borg, and T. A. Baudino, “The dynamics of fibroblast-myocyte-capillary interactions in the heart,” Ann. N. Y. Acad. Sci.1188(1), 143–152 (2010). [CrossRef] [PubMed]

Eur. J. Surg. Oncol.

• E. A. te Velde, T. Veerman, V. Subramaniam, and T. Ruers, “The use of fluorescent dyes and probes in surgical oncology,” Eur. J. Surg. Oncol.36(1), 6–15 (2010). [CrossRef] [PubMed]

IEEE J. Sel. Top. Quantum Electron.

• A. K. Swan, L. A. Moiseev, C. R. Cantor, B. Davis, S. B. Ippolito, W. C. Karl, B. B. Goldberg, and M. S. Unlu, “Toward nanometer-scale resolution in fluorescence microscopy using spectral self-interference,” IEEE J. Sel. Top. Quantum Electron.9(2), 294–300 (2003). [CrossRef]

J. Nucl. Med.

• A. G. T. Terwisscha van Scheltinga, G. M. van Dam, W. B. Nagengast, V. Ntziachristos, H. Hollema, J. L. Herek, C. P. Schröder, J. G. W. Kosterink, M. N. Lub-de Hoog, and E. G. E. de Vries, “Intraoperative near-infrared fluorescence tumor imaging with vascular endothelial growth factor and human epidermal growth factor receptor 2 targeting antibodies,” J. Nucl. Med.52(11), 1778–1785 (2011). [CrossRef] [PubMed]
• L. Sampath, S. Kwon, S. Ke, W. Wang, R. Schiff, M. E. Mawad, and E. M. Sevick-Muraca, “Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer,” J. Nucl. Med.48(9), 1501–1510 (2007). [CrossRef] [PubMed]

J. Opt. Soc. Am. A

• M. Dogan, M. I. Aksun, A. K. Swan, B. B. Goldberg, and M. S. Unlü, “Closed-form representations of field components of fluorescent emitters in layered media,” J. Opt. Soc. Am. A26(6), 1458–1466 (2009). [CrossRef] [PubMed]

Mol. Pharm.

• P. Zou, S. Xu, S. P. Povoski, A. Wang, M. A. Johnson, E. W. Martin, V. Subramaniam, R. Xu, and D. Sun, “Near-infrared fluorescence labeled anti-TAG-72 monoclonal antibodies for tumor imaging in colorectal cancer xenograft mice,” Mol. Pharm.6, 428–440 (2009). [CrossRef] [PubMed]

Nat. Med.

• G. M. van Dam, G. Themelis, L. M. Crane, N. J. Harlaar, R. G. Pleijhuis, W. Kelder, A. Sarantopoulos, J. S. de Jong, H. J. Arts, A. G. van der Zee, J. Bart, P. S. Low, and V. Ntziachristos, “Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results,” Nat. Med.17(10), 1315–1319 (2011). [CrossRef] [PubMed]

Opt. Commun.

• K. E. Drabe, G. Cnossen, and D. A. Wiersma, “Localization of spontaneous emission in front of a mirror,” Opt. Commun.73(2), 91–95 (1989). [CrossRef]

Opt. Express

• A. Bilenca, A. Ozcan, B. Bouma, and G. Tearney, “Fluorescence coherence tomography,” Opt. Express14(16), 7134–7143 (2006). [CrossRef] [PubMed]
• B. Park, M. C. Pierce, B. Cense, S.-H. Yun, M. Mujat, G. Tearney, B. Bouma, and J. de Boer, “Real-time fiber-based multi-functional spectral-domain optical coherence tomography at 1.3 microm,” Opt. Express13(11), 3931–3944 (2005). [CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

• G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A.106(9), 3125–3130 (2009). [CrossRef] [PubMed]

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