Live‐Animal Imaging of Renal Function by Multiphoton Microscopy

Kenneth W. Dunn1, Timothy A. Sutton1, Ruben M. Sandoval1

1 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.9
DOI:  10.1002/0471142956.cy1209s62
Online Posting Date:  October, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high‐resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high‐speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. Curr. Protoc. Cytom. 62:12.9.1‐12.9.18. © 2012 by John Wiley & Sons, Inc.

Keywords: multiphoton microscopy; intravital microscopy; in vivo microscopy; fluorescence microscopy

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Glomerular Permeability
  • Basic Protocol 2: Proximal Tubule Endocytosis
  • Basic Protocol 3: Vascular Flow
  • Basic Protocol 4: Vascular Permeability
  • Basic Protocol 5: Mitochondrial Function
  • Basic Protocol 6: Apoptosis
  • Support Protocol 1: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Glomerular Permeability

  Materials
  • Animal ( protocol 7Support Protocol)
  • 70,000‐Da dextran–Alexa 488 (see recipe)
  • 40,000‐Da dextran‐rhodamine (see recipe)
  • Isotonic saline, sterile
  • 10 mg/ml Hoechst 33342 (Invitrogen)
  • Additional reagents and equipment for animal preparation ( protocol 7)

Basic Protocol 2: Proximal Tubule Endocytosis

  Materials
  • Animal (see protocol 7)
  • 3,000‐Da fluorescent dextran (see recipe), 10,000‐Da fluorescent dextran (see recipe), or fluorescent bovine serum albumin (see recipe)
  • Isotonic saline, sterile
  • Additional reagents and equipment for animal preparation ( protocol 7)

Basic Protocol 3: Vascular Flow

  Materials
  • Animal ( protocol 7)
  • Fluorescent 500,000‐Da dextran (see recipe) or fluorescent bovine serum albumin (see recipe)
  • Additional reagents and equipment for animal preparation ( protocol 7)

Basic Protocol 4: Vascular Permeability

  Materials
  • Animal ( protocol 7)
  • Fluorescent 500,000‐Da dextran (see recipe)
  • Fluorescent 10,000‐Da dextran, 40,000‐Da dextran, 70,000‐Da dextran,150,000‐Da dextran (see recipe), or fluorescent‐labeled bovine serum albumin (see recipe; choice of probe will depend upon the intrinsic permeability of the vessel of interest)
  • Additional reagents and equipment for animal preparation ( protocol 7)

Basic Protocol 5: Mitochondrial Function

  Materials
  • Animal ( protocol 7)
  • 5 mg/ml rhodamine B hexyl ester in dimethylformamide (DMF), anhydrous (store wrapped in foil ≤6 months at −20°C)
  • Isotonic saline, sterile
  • Hoechst 33342 to label nuclei (optional)
  • Additional reagents and equipment for animal preparation ( protocol 7)

Basic Protocol 6: Apoptosis

  Materials
  • Animal ( protocol 7)
  • Hoechst 33342
  • Propidium iodide
  • Isotonic saline, sterile
  • Additional reagents and equipment for preparing the animal ( protocol 7)

Support Protocol 1: Anesthesia and Surgical Creation of a Retroperitoneal Surgical Window for Intravital Imaging

  Materials
  • Animal to be imaged
  • 5% (v/v) and 2% (v/v) isoflurane/oxygen mixtures
  • Pentabarbital (optional)
  • Buprinorphine
  • Germicidal soap
  • 0.9% sterile saline, prewarmed
  • Appropriate probes
  • Anesthesia induction chamber (Braintree Scientific)
  • Homeothermic table (Braintree Scientific)
  • Rectal probe (Braintree Scientific)
  • Electric clippers
  • Vascular catheters (PE‐60 tubing for rats and PE‐50 tubing for mice; Becton Dickinson)
  • Kidney cup
  • Surgical scissors (Braintree Scientific)
  • Appropriate temperature control devices (e.g., circulating water blanket attached to a temperature‐controlled circulating water bath, Repti Therm heating pad)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Brown, E.B., Campbell, R.B., Tsuzuki, Y., Xu, L., Carmeliet, P., Fukumura, D., and Jain, R.K. 2001. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat. Med. 7:864‐868.
   Cahalan, M.D. and Parker, I. 2006. Imaging the choreography of lymphocyte trafficking and the immune response. Curr. Opin. Immunol. 18:476‐482.
   Centonze, V.E. and White, J.G. 1998. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys. J. 75:2015‐2024.
   Condeelis, J. and Segall, J.E. 2003. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer 3:921‐930.
   Denk, W. and Svoboda, K. 1997. Photon upmanship: Why multiphoton imaging is more than a gimmick. Neuron 18:351‐357.
   Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248:73‐76.
   Dunn, K.W. and Young, P.A. 2006. Principles of multiphoton microscopy. Nephron. Exp. Nephrol. 103:e33‐e40.
   Dunn, K.W., Sandoval, R. M., Kelly, K.J., Dagher, P.C., Tanner, G.A., Atkinson, S.J., Bacallao, R.L., and Molitoris, B.A. 2002. Functional studies of the kidney of living animals using multicolor two‐photon microscopy. Am. J. Physiol. Cell Physiol. 283:C905‐C916.
   Ghiron, M. 1912. Uber eine neue Methode mikroskopischer Untersuchung im lebenden Organismus. Zbl. Physiol. 26:613‐617.
   Helmchen, F., Svoboda, K., Denk, W., and Tank, D.W. 1999. In vivo dendritic calcium dynamics in deep‐layer cortical pyramidal neurons. Nat. Neurosci. 2:989‐996.
   Kang, J.J., Toma, I., Sipos, A., McCulloch, F., and Peti‐Peterdi, J. 2006. Quantitative imaging of basic functions in renal (patho)physiology. Am. J. Physiol. Renal Physiol. 291:F495‐F502.
   Kelly, K.J., Sandoval, R.M., Dunn, K.W., Molitoris, B.A., and Dagher, P.C. 2003. A novel method to determine specificity and sensitivity of the TUNEL reaction in the quantitation of apoptosis. Am. J. Physiol. Cell Physiol. 284:C1309‐C1318.
   Kleinfeld, D., Mitra, P.P., Helmchen, F., and Denk, W. 1998. Fluctuations and stimulus‐induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl. Acad. Sci. U.S.A. 95:15741‐15746.
   Konig, K. 2000. Multiphoton microscopy in life sciences. J. Microsc. 200:83‐104.
   Majewska, A., Yiu, G., and Yuste, R. 2000. A custom‐made two‐photon microscope and deconvolution system. Pflugers Arch. 441:398‐408.
   Masters, B.R., So, P.T., and Gratton, E. 1997. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophys. J. 72:2405‐2412.
   Miller, M.J., Wei, S.H., Cahalan, M.D., and Parker, I. 2003. Autonomous T cell trafficking examined in vivo with intravital two‐photon microscopy. Proc. Natl. Acad. Sci. U.S.A. 100:2604‐2609.
   Molitoris, B.A. and Sandoval, R.M. 2005. Intravital multiphoton microscopy of dynamic renal processes. Am. J. Physiol. Renal Physiol. 288:F1084‐F1089.
   Muller, M., Schmidt, J., Mironov, S.L., and Richter, D.W. 2003. Construction and performance of a custom‐built two‐photon laser scanning system. J. Phys. D: Appl. Phys. 36:1747‐1757.
   Nguyen, Q.T., Callamaras, N., Hsieh, C., and Parker, I. 2001. Construction of a two‐photon microscope for video‐rate Ca(2+) imaging. Cell Calcium 30:383‐393.
   Ogasawara, Y., Takehara, K., Yamamoto, T., Hashimoto, R., Nakamoto, H., and Kajiya, F. 2000. Quantitative blood velocity mapping in glomerular capillaries by in vivo observation with an intravital videomicroscope. Methods Inf. Med. 39:175‐178.
   Rosivall, L., Mirzahosseini, S., Toma, I., Sipos, A., and Peti‐Peterdi, J. 2006. Fluid flow in the juxtaglomerular interstitium visualized in vivo. Am. J. Physiol. Renal Physiol. 291:F1241‐F1247.
   Russo, L.M., Sandoval, R.M., McKee, M., Osicka, T.M., Collins, A.B., Brown, D., Molitoris, B.A., and Comper, W.D. 2007. The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: Retrieval is disrupted in nephrotic states. Kidney Int. 71:504‐513.
   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.
   Squirrell, J.M., Wokosin, D.L., White, J.G., and Bavister, B.D. 1999. Long‐term two‐photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol. 17:763‐767.
   Steinhausen, M. and Tanner, G.A. 1976. Microcirculation and tubular urine flow in the mammalian kidney cortex (in vivo microscopy). Sitzungsberichte Heidelberger Akad Wissensch Math‐naturwissensch Kl 3:279‐335.
   Steinhausen, M., Eisenbach, G.M., and Bottcher, W. 1973. High‐frequency microcinematographic measurements on peritubular blood flow under control conditions and after temporary ischemia of rat kidneys. Pflugers Arch. 339:273‐288.
   Sutton, T.A., Mang, H.E., Campos, S.B., Sandoval, R.M., Yoder, M.C., and Molitoris, B.A. 2003. Injury of the renal microvascular endothelium alters barrier function after ischemia. Am. J. Physiol. Renal Physiol. 285:F191‐F198.
   Sutton, T.A., Kelly, K.J., Mang, H.E., Plotkin, Z., Sandoval, R.M., and Dagher, P.C. 2005. Minocycline reduces renal microvascular leakage in a rat model of ischemic renal injury. Am. J. Physiol. Renal Physiol. 288:F91‐F97.
   Svoboda, K. and Yasuda, R. 2006. Principles of two‐photon excitation microscopy and its applications to neuroscience. Neuron 50:823‐839.
   Svoboda, K., Denk, W., Kleinfeld, D., and Tank, D.W. 1997. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385:161‐165.
   Tanner, G.A., Sandoval, R.M., and Dunn, K.W. 2004. Two‐photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys. Am. J. Physiol. Renal Physiol. 286:F152‐F160.
   te Velde, E.A., Reijerkerk, A., Brandsma, D., Vogten, J.M., Wu, Y., Kranenburg, O., Voest, E.E., Gebbink, M., and Borel Rinkes, I.H. 2005. Early endostatin treatment inhibits metastatic seeding of murine colorectal cancer cells in the liver and their adhesion to endothelial cells. Br. J. Cancer 92:729‐735.
   Toma, I., Kang, J.J., and Peti‐Peterdi, J. 2006. Imaging renin content and release in the living kidney. Nephron. Physiol. 103:71‐74.
   Watson, A.J., Chu, S., Sieck, L., Gerasimenko, O., Bullen, T., Campbell, F., McKenna, M., Rose, T., and Montrose, M.H. 2005. Epithelial barrier function in vivo is sustained despite gaps in epithelial layers. Gastroenterology 129:902‐912.
   Yamamoto, T., Tada, T., Brodsky, S.V, Tanaka, H., Noiri, E., Kajiya, F., and Goligorsky, M.S. 2002. Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am. J. Physiol. Renal Physiol. 282:1150‐1155.
   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. 289:1197‐1208.
   Zipfel, W.R., Williams, R.M., and Webb, W.W. 2003. Nonlinear magic: Multiphoton microscopy in the biosciences. Nat. Biotechnol. 21:1369‐1377.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library