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

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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‐HCO₃ solution (see recipe )
Experimental solution
PBS ( appendix 2A )
MgCl₂
CaCl₂
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‐HCO₃ 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% O₂ /5% CO₂ source
Control tubular perfusate (see recipe )
Krebs Ringer‐HCO₃ 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 a a	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

^a TMA‐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‐HCO₃ (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

Figure 12.12.1 Fluorescence imaging of primary cultures and cell lines to study morphology and compartments. DIC (A ) and fluorescence (B ) images of primary vascular smooth muscle cells derived from manually dissected arteriolar explants. (C ) Quinacrine‐stained renin granules in As 4.1 cells, a renin‐secreting tumor cell line. (D ) Renal medullary interstitial cells in culture labeled with Nile‐Red staining of lipid vesicles. (E ) A mouse macula densa‐derived cell line is co‐labeled with Mito‐Tracker Red (mitochondria) and quinacrine (green, acidic granules).

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Figure 12.12.2 Spectrofluorometry readings for ratiometric calcium signaling (A ) and a renin‐FRET enzymatic activity assay (B ). (A ) Representative recording and procedure of calibrating fura‐2 fluorescence into absolute values of [Ca²⁺ ]. The emission spectrum collected from 380 nm excitation (gray) and the intracellular calcium ratio (black) are used to calculate absolute concentrations according to the equation described in the methods. (B ) The increase in slope shown across the time duration of the arrow (initial rate) corresponds to an increase in EDANS fluorescence (ANG I production) due to cleavage of the substrate from renin enzymatic activity.

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Figure 12.12.3 Imaging in vitro preparations microdissected from mouse kidney. (A ) A transmitted light differential interference contrast (DIC) image demonstrating the afferent arteriole (AA) and attached glomerulus (G). (B ) Fluorescence image of an AA‐G preparation. Renin granules (green) are labeled with quinacrine, and plasma membrane (red) is labeled with R18. (C ) Fluorescence image of a glomerulus using the nuclear stain DAPI (blue) and the membrane‐stain TMA‐DPH to label the podocytes found surrounding glomerular endothelial cells. (D ) DIC image of a glomerulus with afferent arteriole (AA) and attached tubule segment (cTAL) for double perfusion studies. Fluorescence image with quinacrine‐stained renin granules (green) is superimposed. (E ) Pseudocolor image of an in vitro afferent arteriole‐glomerulus preparation stained with the Stark‐shift voltage sensitive dye, annine‐6, showing variations in membrane potential in individual vascular smooth muscle cells.

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Figure 12.12.4 In vivo imaging and quantification of renal functional parameters in a Munich‐Wistar rat. Quinacrine (green) is used to label acidic compartments (including renin granules) and 70‐kDa rhodamine (red) marks plasma in the intravascular space. (A ) Various cortical segments of the nephron may be visualized by z‐sectioning down to 200 µm below the surface of the kidney. A glomerulus (G) opens up into the proximal tubule (PT), and a collecting duct (CD) lies adjacent. Multiple functional compartments may be visualized simultaneously, down to the subcellular level. (B ) The single nephron glomerular filtration rate (SNGFR) may be measured taking xy‐t video recordings and calculating the flow of Lucifer Yellow, an extracellular fluid marker, down the early portion of the PT. (C ) In vivo imaging of intracellular pH in the proximal tubule (PT). BCECF‐AM was loaded under the renal capsule in the living kidney to measure cell pH. Note the primarily apical, microvillar BCECF fluorescence in the PT indicating alkalotic conditions (bicarbonate reabsorption).

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Literature Cited

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