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Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching

Nature Methods volume 3, pages 275–280 (2006)Cite this article

Abstract

It has not been possible to measure diffusion deep in solid tissues such as tumors because of the limited light penetration of conventional optical techniques. Here we report a microfiberoptic epifluorescence photobleaching (MFEP) method in which photobleaching is done by laser epi-illumination through a multimode fiberoptic whose micron-sized tip can be introduced deep into tissues. We applied MFEP to measure the diffusion of fluorescent macromolecules in tumors in living mice, at depths well beyond those accessible by surface optical measurements. Macromolecule diffusion was slowed about twofold within 200 μm of the surface of a solid tumor, but was slowed greater than tenfold beyond 500 μm. Our results reveal a remarkable and previously unrecognized slowing of diffusion deep in tumors, which correlated with the differing tissue architectures of tumor periphery versus core, and with altered tumor vasculature produced by aquaporin-1 deletion. MFEP should have wide applications for measuring diffusion in organs, solid tumors and other light-inaccessible tissue masses.

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Figure 1: Apparatus and in vitro validation of MFEP.
Figure 2: Determination of diffusion coefficients from MFEP recovery data.
Figure 3: Diffusion in collagen and gelatin gels.
Figure 4: Diffusion in solid tumor.

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Acknowledgements

Supported by grants EB00415, DK35124, EY13574, HL59198, DK72517 and HL73856 from the US National Institutes of Health, and a Research Development Program grant from the Cystic Fibrosis Foundation.

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Author notes

  1. Jay R Thiagarajah and Jung Kyung Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, 94143, California, USA

    Jay R Thiagarajah, Jung Kyung Kim, Mazin Magzoub & A S Verkman

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  1. Jay R Thiagarajah
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  2. Jung Kyung Kim
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  3. Mazin Magzoub
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  4. A S Verkman
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Correspondence to A S Verkman.

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Supplementary information

Supplementary Fig. 1

Comparison of simulated recovery curves with analytical solution for the case of three-dimensional photobleaching in isotropic media. (PDF 33 kb)

Supplementary Fig. 2

Log-log plot of theoretically predicted fluorescence recovery half-times as a function of D/D0. (PDF 17 kb)

Supplementary Fig. 3

Simulated fluorescence recovery curves for diffusion with convection around the fiberoptic with different tip diameters. (PDF 30 kb)

Supplementary Fig. 4

Theoretically predicted half-times from the simulated fluorescence recovery of 10 kDa FITC-dextran in solution. (PDF 18 kb)

Supplementary Methods (PDF 32 kb)

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Thiagarajah, J., Kim, J., Magzoub, M. et al. Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching. Nat Methods 3, 275–280 (2006). https://doi.org/10.1038/nmeth863

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