From In Vitro to In Vivo: Imaging from the Single Cell to the Whole Organism

Jung Julie Kang1, Ildiko Toma1, Arnold Sipos1, Janos Peti‐Peterdi1

1 University of Southern California, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.12
DOI:  10.1002/0471142956.cy1212s44
Online Posting Date:  April, 2008
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Abstract

This unit addresses the applications of fluorescence microscopy and quantitative imaging to study multiple physiological variables of living tissue. Protocols are presented for fluorescence‐based investigations ranging from in vitro cell and tissue approaches to in vivo imaging of intact organs. These include the measurement of cytosolic parameters both in vitro and in vivo (such as calcium, pH, and nitric oxide), dynamic cellular processes (renin granule exocytosis), FRET‐based real‐time assays of enzymatic activity (renin), physiological processes (vascular contraction, membrane depolarization), and whole organ functional parameters (blood flow, glomerular filtration). Multi‐photon microscopy is ideal for minimally invasive and undisruptive deep optical sectioning of the living tissue, which translates into ultra‐sensitive real‐time measurement of these parameters with high spatial and temporal resolution. With the combination of cell and tissue cultures, microperfusion techniques, and whole organ or animal models, fluorescence imaging provides unmatched versatility for biological and medical studies of the living organism. Curr. Protocol. Cytom. 44:12.12.1‐12.12.26. © 2008 by John Wiley & Sons, Inc.

Keywords: in vivo imaging; in vitro imaging; multiphoton fluorescence microscopy; real‐time imaging; intravital imaging; laser‐scanning microscopy

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Fluorescence Studies of Cultured Cells: Cell Signaling Studies, Primary Culture, or Cell Lines
  • Basic Protocol 1: Cuvette‐Based Spectrofluorometry to Assess Second‐Messenger Signaling in Living Cells
  • Support Protocol 1: Enhanced Calcium Loading Techniques with Fura‐2 and Fluo‐4
  • Basic Protocol 2: Cuvette‐Based Spectrofluorometry to Assess Nitric Oxide Production
  • Basic Protocol 3: A Novel Application of FRET: Cuvette‐Based Spectrofluorometry to Evaluate Cellular Enzyme Activity
  • Basic Protocol 4: In Vitro Tissue Imaging of Renin Release: Isolated Microperfused Tissue, JGA, Renal Medulla
  • Basic Protocol 5: Quantitative Imaging of Kidney Functions In Vivo by Multiphoton Excitation Laser Scanning Fluorescence Microscopy
  • Basic Protocol 6: In Vivo Imaging of Cytosolic Parameters (Ca2+, pH)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cuvette‐Based Spectrofluorometry to Assess Second‐Messenger Signaling in Living Cells

  Materials
  • Cells of interest
  • Fura‐2 ratiometric calcium imaging dye (Invitrogen)
  • Dimethyl sulfoxide (DMSO)
  • Krebs Ringer‐HCO 3 solution (see recipe)
  • Experimental solution
  • PBS ( appendix 2A)
  • MgCl 2
  • CaCl 2
  • Ionomycin (membrane permeabilizer)
  • 24 × 40–mm glass coverslips
  • Cuvette‐based spectrofluorometer (Quantamaster‐8, Photon Technology) with heated cuvette holder block (37°C) and quartz cuvettes
  • Peristaltic pump, vacuum, polyethylene tubing for cuvette superfusion and fluid exchange

Support Protocol 1: Enhanced Calcium Loading Techniques with Fura‐2 and Fluo‐4

  Materials
  • Endothelial cells of interest
  • DAF‐FM diacetate (nitric oxide imaging dye; Invitrogen)
  • Dimethyl sulfoxide (DMSO)
  • Krebs Ringer‐HCO 3 solution (see recipe), 37°C
  • Sodium nitroprusside (SNP; Sigma)
  • Glass coverslips
  • Cuvette‐based spectrofluorometer (Quantamaster‐8, Photon Technology) with heated cuvette holder block (37°C) and quartz cuvettes

Basic Protocol 2: Cuvette‐Based Spectrofluorometry to Assess Nitric Oxide Production

  Materials
  • Male mice (for fresh kidney tissue; C57Bl/6, 20 g, 6 to 8 weeks old)
  • Inactin (see recipe)
  • Protease inhibitor (BD Biosciences)
  • Tissue homogenization buffer (see recipe)
  • Renin assay buffer (see recipe)
  • Renin‐FRET substrate (AnaSpec; see recipe)
  • Tissue homogenizer (Ultra‐Turrax T25 basic, IKA)
  • Orbital shaker
  • 1‐ml microcentrifuge tubes
  • Cuvette‐based spectrofluorometer (Quantamaster‐8, Photon Technology)

Basic Protocol 3: A Novel Application of FRET: Cuvette‐Based Spectrofluorometry to Evaluate Cellular Enzyme Activity

  Materials
  • Mice (15 to 20 g)
  • Inactin (see recipe)
  • Dissection medium (see recipe)
  • Bath medium (see recipe)
  • 95% O 2/5% CO 2 source
  • Control tubular perfusate (see recipe)
  • Krebs Ringer‐HCO 3 solution (see recipe)
  • Fluorescent dyes of interest (e. g., quinacrine)
  • Thermoregulated Lucite chamber (Vestavia Scientific)
  • Confocal microscope system (e. g., Leica TCS SP2, Leica Microsystem)
  • Glass pipets (35‐µm o. d.)
  • Imaging software (e. g., Leica LCS)
NOTE: Table 12.12.2 provides a list of fluorophores used to specifically label various structures or fields of interest within the afferent arteriole‐glomerulus complex. Excitation/emission parameters and dye loading recommendations are also included.
Table 2.2.2   MaterialsFluorescent Probes Commonly Used in In Vitro Experiments

Dye aa Target One photon/multiphoton excitation (nm) Loading
TMA‐DPH Cell membrane 375/755 1 mM perfusate
R18 Cell membrane 543/800 2 mM perfusate
Hoechst 33342 Nucleus 380/760 2 mM perfusate/bath
Quinacrine Acidic granules (renin) 290/860 5 mM perfusate/bath
Lyso Tracker Red Acidic granules (renin) 598 5 mM perfusate
Renin‐FRET substrate (EDANS) Renin enzymatic activity 360/720 2 mM bath
Fluo 4 Intracellular calcium 488/850 10 mM perfusate/bath
Fura Red Intracellular calcium (↑ ratio = ↑ Ca2+) 488/850 10 mM perfusate/bath
DAF‐FM Nitric oxide 495 10 mM perfusate/bath
BCECF pH (↑ ratio = ↑ pH) 495/440 10 mM perfusate/bath
Nile Red Neutral lipids 543/800 2 mM bath

 aTMA‐DPH, 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐1,3,5‐hexatriene p‐toluenesulfonate; EDANS, 5‐(2‐aminoethylamino) nephthalone‐1‐sulfonic acid. All fluorophores are available from Molecular Probes, except quinacrine (Sigma) and renin substrate (DABCYL‐ ‐Abu‐Ile‐His‐Pro‐Phe‐His‐Leu‐Val‐Ile‐His‐Thr‐EDANS, AnaSpec).

Basic Protocol 4: In Vitro Tissue Imaging of Renin Release: Isolated Microperfused Tissue, JGA, Renal Medulla

  Materials
  • C57 BL/6 mice or Munich‐Wistar rats
  • Inactin (see recipe)
  • Ketamine
  • Krebs Ringer‐HCO 3 (see recipe)
  • Fluorescent probes (70‐kDa rhodamine B conjugate, quinacrine, Lucifer Yellow)
  • Polyethylene tubing (0.86‐mm i. d.)
  • Analog single‐channel transducer signal conditioner (World Precision Instruments model no. BP‐1)
  • Two‐photon laser scanning fluorescence microscope (e. g., Leica TCS SP2 AOBS MP confocal microscope system) and HCX PL APO 63×/1.4NA oil CS objective lens (Leica)
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Figures

Videos

Literature Cited

Literature Cited
   Denk, W., Strickler, J. H., and Webb, W. W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248: 73‐76.
   Garaschuk, O., Milos, R.I., and Konnerth, A. 2006. Targeted bulk‐loading of fluorescent indicators for two‐photon brain imaging in vivo. Nat. Protoc. 1: 380‐386.
   Hwang, E. F., Williams, I., Kovacs, G., Peti‐Peterdi, J., Siroky, B., Rice, W. C., Bates, E., Schweibert, E.M., Unlap, M. T., and Bell, P.D. 2003. Impaired ability of the Na+/ Ca2+ exchanger from the Dahl/Rapp salt‐sensitive rat to regulate cytosolic calcium. Am. J. Physiol. Renal Physiol. 284: F1023‐F1031.
   Kang, J. J., Toma, I., Sipos, A., McCulloch, F., and Peti‐Peterdi, J. 2006a. Quantitative imaging of basic functions in renal (patho)physiology. Am. J. Physiol. Renal Physiol. 291(2): F495‐F502.
   Kang, J. J., Toma, I., Sipos, A., McCulloch, F., and Peti‐Peterdi, J. 2006b. Imaging the renin‐angiotensin system: An important target of anti‐hypertensive therapy. Adv. Drug Deliv. Rev. 58: 824‐833.
   Komlosi, P., Fintha, A., and Bell, P.D. 2006. Unraveling the relationship between macula densa cell volume and luminal solute concentration/osmolality. Kidney Int. 70: 865‐871.
   Kovacs, G., Komlosi, P., Fuson, A., Peti‐Peterdi, J., Rosivall, L., and Bell, P.D. 2003. Neuronal nitric oxide synthase: Its role and regulation in macula densa cells. J. Am. Soc. Nephrol. 14: 2475‐2483.
   McDonald, D.M. and Choyke, P. L. 2003. Imaging of angiogenesis: From microscope to clinic. Nat. Med. 9: 713‐725.
   Peti‐Peterdi, J. 2006. Calcium wave of tubuloglomerular feedback. Am. J. Physiol. Renal Physiol. 291: F473‐F480.
   Peti‐Peterdi, J. and Bell, P.D. 1999. Cytosolic [Ca2+] signaling pathway in macula densa cells. Am. J. Physiol. 277: F472‐F476.
   Peti‐Peterdi, J., Chambrey, R., Bebok, Z., Biemesderfer, D., St. John, P. L., Abrahamson, D.R., Warnock, D.G., and Bell, P.D. 2000. Macula densa Na(+)/H(+) exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms. Am. J. Physiol. Renal Physiol. 278: F452‐F463.
   Peti‐Peterdi, J., Morishima, S., Bell, P.D., and Okada, Y. 2002a. Two‐photon excitation fluorescence imaging of the living juxtaglomerular apparatus. Am. J. Physiol. Renal Physiol. 283: F197‐F201.
   Peti‐Peterdi, J., Bebok, Z., Lapointe, J.Y., and Bell, P.D. 2002b. Novel regulation of cell [Na(+)] in macula densa cells: Apical Na(+) recycling by H‐K‐ATPase. Am. J. Physiol. Renal Physiol. 282: F324‐F329.
   Peti‐Peterdi, J., Komlosi, P., Fuson, A. L., Guan, Y., Schneider, A., Qi, Z., Redha, R., Rosivall, L., Breyer, M.D., and Bell, P.D. 2003. Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells. J. Clin. Invest. 112: 76‐82.
   Peti‐Peterdi, J., Fintha, A., Fuson, A. L., Tousson, A., and Chow, R. H. 2004. Real‐time imaging of renin release in vitro. Am. J. Physiol. Renal Physiol. 287: F329‐F335.
   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.
   Sipos, A., Toma, I., Kang, J. J., Rosivall, L., and Peti‐Peterdi, J. 2007. Advances in renal (patho)physiology using multiphoton microscopy. Kidney Int. 72: 1188‐1191.
   St. Croix, C.M., Leelavanichkul, K., and Watkins, S. C. 2006. Intravital fluorescence microscopy in pulmonary research. Adv. Drug Deliv. Rev. 58: 834‐840.
   Svoboda, K., Helmchen, F., Denk, W., and Tank, D. W. 1999. Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo. Nat. Neurosci. 2: 65‐73.
   Toma, I., Kang, J. J., and Peti‐Peterdi, J. 2006. Imaging renin content and release in the living kidney. Nephron. Physiol. 103: 71‐74.
   Wang, G. T., Chung, C. C., Holzman, T. F., and Krafft, G. A. 1993. A continuous fluorescence assay of renin activity. Anal. Biochem. 210: 351‐359.
   Wang, W., Wyckoff, J.B., Frohlich, V. C., Oleynikov, Y., Hüttelmaier, S., Zavadil, J., Cermak, L., Bottinger, E.P., Singer, R. H., White, J.G., Segall, J.E., and Condeelis, J. S. 2002. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res. 62: 6278‐6288.
   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.
   Yuste, R. and Denk, W. 1995. Dendritic spines as basic functional units of neuronal integration. Nature. 375: 682‐684.
   Zsembery, A., Boyce, A. T., Liang, L., Peti‐Peterdi, J., Bell, P.D., and Schwiebert, E.M. 2003. Sustained calcium entry through P2′ nucleotide receptor channels in human airway epithelial cells. J. Biol. Chem. 278: 13398‐13408.
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