Summary
The recent introduction of multiphoton microscopy coupled with advances in optics, computer sciences, designer fluorophores, molecular labeling, and previously developed physiologic approaches have empowered investigators to quantitatively study the cell-specific dynamic events, such as endocytosis, within a functioning organ with subcellular resolution. This rapidly emerging field of investigation, with superior spatial and temporal resolution and high sensitivity, enables investigators to track molecules and determine their mode of cellular uptake, intracellular trafficking, and metabolism in a cell-specific fashion in complex heterogeneous organs such as the kidney with repeated determinations possible over a prolonged period of time. This approach is enhanced by the ability to obtain and quantify volumetric data with using up to three different fluorophores simultaneously. We have utilized this intravital approach to understand and quantify kidney proximal tubule cell uptake and intracellular distribution and metabolism of fluorescently labeled molecules, including folic acid, gentamicin, and small interfering ribonucleic acid (siRNA). Limitations of this technique include tissue penetration, which is the major barrier to successful clinical utilization of this technology. However, its use in preclinical animal models offers new insight into physiologic processes and the pathophysiology and treatment of disease processes.
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References
1. Sandoval, R.M., Kennedy, M.D., Low, P.S., and Molitoris, B.A. (2004). Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy. Am. J. Physiol. Cell Physiol. 287, C517–C526.
2. Molitoris, B.A., and Sandoval, R.M. (2005). Intravital multiphoton microscopy of dynamic renal processes. Am. J. Physiol. Renal Physiol. 288, F1084–F1089.
3. Molitoris, B.A., and Sandoval, R.M. (2006). Pharmacophotonics: utilizing multi-photon microscopy to quantify drug delivery and intracellular trafficking in the kidney. Adv. Drug Deliv. Rev. 58, 809–823.
4. Sandoval, R.M., Reilly, J.P., Running, W., et al. (2006). A non-nephrotoxic gentamicin congener that retains antimicrobial efficacy. J. Am. Soc. Nephrol. 17, 2697–2705.
5. Yu, W., Sandoval, R.M., and Molitoris, B.A. (2005). Quantitative intravital microscopy using a generalized polarity concept for kidney studies. Am. J. Physiol. Cell Physiol. 289, C1197–C1208.
6. Dunn, K.W., Sandoval, R.M., Kelly, K.J., et al. (2002). Functional studies of the kidney of living animals using multicolor two-photon microscopy. Am. J. Physiol. Cell Physiol. 283, C905–C916.
Acknowledgements
We are indebted to Kenneth W. Dunn for invaluable input regarding ongoing studies. This work was supported by National Institutes of Health grants P50-DK-61594 and PO1-DK-53465, a Veterans Affairs Merit Review (to B.A. Molitoris), and an Indiana Genomics Initiative grant from the Lilly Foundation to the Indiana University School of Medicine.
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Sandoval, R.M., Molitoris, B.A. (2008). Quantifying Endocytosis In Vivo Using Intravital Two-Photon Microscopy. In: Ivanov, A.I. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 440. Humana Press. https://doi.org/10.1007/978-1-59745-178-9_28
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DOI: https://doi.org/10.1007/978-1-59745-178-9_28
Publisher Name: Humana Press
Print ISBN: 978-1-58829-865-2
Online ISBN: 978-1-59745-178-9
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