IMAGING From Single Cell to the Whole Organism

UNIT 12.12From In Vitro to In Vivo: Imaging fromthe Single Cell to the Whole OrganismJung Julie Kang,1 Ildiko Toma,1 Arnold Sipos,1 and Janos Peti-Peterdi11University of Southern California, Los Angeles, California

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

Keywords: in vivo imaging in vitro imaging multiphoton fluorescencemicroscopy real-time imaging intravital imaging laser-scanning microscopy

INTRODUCTIONThe application of in vitro fluorescence imaging to cellular studies permits importantdiscoveries about structure, function, responses to the environment, intracellular signal-ing pathways, and, indirectly, intercellular relationships. Such studies are particularlyuseful in determining the specific mechanisms by which particular cellular componentscontribute to larger processes, such as the roles of endothelial nitric oxide productionin vasodilation or the relevance of vascular smooth muscle calcium concentration tovasoconstriction. Furthermore, both the acute and chronic effects of various stimuli maybe investigated: varying the culturing media or conditions of cells may cause changes incell signaling or function, which can be detected with quantitative imaging. Fluorescenceimaging has tremendous applicability to studies of cytosolic changes (e.g., calcium, pH,cell volume), cellular responses to stimuli (e.g., nitric oxide, prostaglandins), and proteincontent/enzyme activity (e.g., renin). Cellular studies permit the determination of themechanisms responsible for propagating complex pathways, providing valuable targetsfor intervention, especially in disease.

This unit delineates several different protocols for applying fluorescence imaging technol-ogy to cellular studies. The superior image quality and resolution permits the visualizationof morphology in living cells, as shown with primary cell cultures of vascular smoothmuscle in Figure 12.12.1 A,B. The acidophilic fluorophore, quinacrine, may be usedto label renin granules in appropriate cells, shown in Figure 12.12.1 C. Lipid vesiclesmay be identified with the red stain, Nile Red, shown in Figure 12.12.1D. The judiciousselection of dyes permits simultaneous co-labeling of multiple intracellular structures,

Current Protocols in Cytometry 12.12.1-12.12.26, April 2008Published online April 2008 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142956.cy1212s44Copyright C 2008 John Wiley & Sons, Inc.

Cellular andMolecularImaging

12.12.1Supplement 44

From In Vitro toIn Vivo: Imagingfrom the Single

Cell to the WholeOrganism

12.12.2Supplement 44 Current Protocols in Cytometry

Figure 12.12.1 Fluorescence imaging of primary cultures and cell lines to study morphologyand compartments. DIC (A) and uorescence (B) images of primary vascular smooth musclecells derived from manually dissected arteriolar explants. (C) Quinacrine-stained renin granules inAs 4.1 cells, a renin-secreting tumor cell line. (D) Renal medullary interstitial cells in culture labeledwith Nile-Red staining of lipid vesicles. (E) A mouse macula densa-derived cell line is co-labeledwith Mito-Tracker Red (mitochondria) and quinacrine (green, acidic granules). For a color versionof this gure, see http://www.currentprotocols.com.


12.12.3Current Protocols in Cytometry Supplement 44

Table 12.12.1 Dyes Used in Cellular Cuvette-Based SpectrouorometryStudies

Dye Parameter measured Excitation Emission

Renin-FRETSubstrate

Renin enzymatic activity 340 nm 490 nm

Fura-2 Intracellular calcium( ratio = Ca2+)

340/380 nm 510 nm

Fluo 4 Intracellular calcium 488 nm 516 nmDAF-FM Nitric oxide 495 nm 515 nmBCECF pH ( ratio = pH) 500/440 nm 530 nm

and the use of Mito-Tracker Red for mitochondria and quinacrine for acidic granules inmacula densa cells beautifully illustrates the morphology and highlights the specificityof each of these dyes for their organelles (Fig. 12.12.1E). The imaging system may alsobe used to visualize fixed cells for immunocytochemistry experiments. In addition tostructural characterization, the appropriate selection of fluorescent probes permits theapplication of cuvette-based spectrofluorometry to investigate direct cause-and-effectrelationships by studying cytosolic signals and second messengers in response to dif-ferent stimuli. Table 12.12.1 lists various cellular structural and messenger dyes usefulfor spectrofluorometry studies. Quantification of cellular enzymatic activity is anothervaluable application of this technique. A recently developed fluorogenic peptide based onfluorescence resonance energy transfer (FRET) permits real-time measurements of reninactivity and can be used to analyze enzymatic activity of tissue samples from healthy anddiseased animals. Imaging studies on cells have limitless applicability to studying directand acute effects as well as evaluating changes in chronic disease conditions.

STRATEGIC PLANNINGCuvette-Based Spectrouorometry to Assess Second-Messenger Signalingin Living CellsSignal transduction refers to the process by which a cell receives input and convertsit to another signal, typically involving an ordered sequence of intracellular reactionscarried out by enzymes and linked through second messengers. The association of stim-ulus with signal transduction pathway, second messenger, and ultimately, end-response,provides important mechanistic information that can be utilized to promote favorableand inhibit detrimental processes. Stimuli that initiate a cellular response may be molec-ular (hormones, cytokines), environmental (extracellular matrix), or physical (light) innature. Some cellular responses to extracellular stimulation that depend on signal trans-duction include metabolism and cell proliferation/death. Therefore, the translation ofexternal cues into internal messages that elicit specific cellular actions involves signaltransduction. Many diverse disease processes including diabetes, hypertension, autoim-munity, and cancer arise from defects in signal transduction pathways, elucidating theimportance of signal transduction to physiology as well as pathology.

In vitro cell signalingFluorescence microscopy has vast applicability to cell signaling studies. Alterations inpH (Peti-Peterdi et al., 2000), sodium (Peti-Peterdi et al., 2002a), and nitric oxide (Kovacset al., 2003) have all been investigated with commercially available probes. Fluorescenceimaging can also be used to study intracellular changes in response to different stimuli.Signals such as hormones and growth factors are received at cell surface receptors andthen relayed to target molecules in the cytosol and/or nucleus by second messenger




molecules. In addition to functioning as molecules that translate extracellular cues intointracellular messages, second messengers greatly increase the amplitude of the signal.Major classes of second messengers include cyclic nucleotides (e. g., cAMP and cGMP),inositol trisphosphate (IP3), diacylglycerol (DAG), and calcium ions. Fluorescent dyesfor many second messengers exist, so prudent experimental design can provide definitiveconfirmation of the effects of hypothesized inputs on cellular behavior. For example,the technology may be applied to investigating the effects of a proposed substrate ona G proteincoupled receptormediated intracellular calcium signal. Furthermore, theeffects of receptor inhibition on the second messenger signal may be analyzed with thisapproach. Ultimately, the data obtained can elucidate the influence of a stimulus on acell by assessing changes in calcium signaling in larger physiological or pathologicalprocesses (Peti-Peterdi et al., 2002a).

Calcium signalingSince intracellular signal transduction is largely carried out by second messengermolecules, identification of changes in second messengers provides evidence of a di-rect effect of stimulus on cell behavior. Calcium is one of the most widely studied secondmessengers because it is used in a multitude of processes, including muscle contraction,the release of neurotransmitters, cell proliferation, secretion, cytoskeletal management,cell movement, gene expression, and metabolism. Intracellular calcium concentrationis normally maintained at very low levels by sequestration in the smooth endoplasmicreticulum and mitochondria. Three main signals that promote the activation and releaseof calcium are G proteincoupled receptor-regulated pathways, receptor tyrosine kinasepathways, and ligand or current-gated ion channels. Its release from the endoplasmicreticulum results in its binding to and activation of proteins or enzymes.

The first dye to be highly used for calcium imaging was Fura-2, a ratiometric fluorescentdye that binds to free intracellular calcium. Its fluorescence is detected at 510 nm inresponse to alternate excitation at 340 and 380 nm, with the ratio of the two (340/380) di-rectly correlating to the amount of intracellular calcium. The use of a ratio resolves someexperimental challenges such as dye concentration and background autofluorescence,making it an ideal choice for quantitative measurements. Fura-2 is thus the preferen-tial dye for ratiometric calcium imaging when the alternation of excitation wavelengthsis more practical than the detection of multiple emission wavelengths. A newer gen-eration of calcium fluorophores includes fluo-4, which provides an efficient, validatedmethod of imaging intracellular calcium fluxes. Because fluo-4 AM loads faster andoffers greater fluorescence at equivalent concentrations, it is the preferred indicator forconfocal microscopy, flow cytometry, and microplate screening applications. Althougheach fluorescent calcium probe has different benefits, their combined use may providean extra level of validation of the data obtained. Fluo-4 and Fura Red respond to [Ca2+]ichanges with no significant kinetic differences. However, fluo-4 fluorescence increaseswith a rise in [Ca2+]i, while Fura Red fluorescence decreases. These features make Fluo-4and Fura Red an excellent dye pair for ratiometric [Ca2+]i imaging (Peti-Peterdi, 2006).The application and choice of various fluorophores depends on the fluorescence equip-ment available and on the biological processes (range of calcium changes) to be examined.Ratiometric approaches (the simultaneous use of two different dyes or dye forms thathave similar loading characteristics) are almost always recommended, which excludethe possibility of artifacts associated with dye loading, photobleaching, leakage, cellvolume changes, etc. For the widely used xenon light source-based systems, fura-2 isan ideal choice since excitation ratiometric approaches can be used. For instrumentspowered by laser sources, such as confocal and multi-photon fluorescence microscopes,emission ratiometric approaches are typically required because of the single, fixed ex-citation wavelength. For these systems, the fluo-4/Fura Red ratio pair is the preferred



selection for calcium imaging. Fluo-4 and Fura Red AM forms (cell membrane perme-able for loading) are non-fluorescent, as opposed to fura-2 and indo-1, and do not tendto compartmentalize, factors that would significantly limit the sensitivity of detectingcytosolic-free calcium. It is recommended to check the calcium dissociation constant(Kd) of different calcium probes, which will give an idea about the range of calcium thatthe given fluorophore can detect. For example fura-2 (Kd = 224 nM) and fluo-4 (Kd =345 nM) are used to detect low or medium cytosolic calcium levels while fluo-5 F is thechoice for high calcium conditions (Kd = 2.3 M).

A Novel Application of FRET: Cuvette-Based Spectrouorometry to EvaluateCellular Enzyme ActivityCuvette-based spectrofluorometry is valuable for investigations that aim to determinethe presence or absence of a direct, acute effect of a given intervention on cellularfunction. In certain circumstances, the magnitude of the effect may even be quantified.Cuvette-based investigations are readily applicable for the study of specific causes andeffects in isolated cells, providing definitive information about external influences or thecellular machinery involved. For example, the cuvette system has been applied to studythe potential value of purinergic receptormediated calcium signaling as a potentialrescue for epithelial cells in cystic fibrosis (Zsembery et al., 2003). Alternatively, theexperimental approach has been used to assess intact cellular machinery by comparingchanges in second messenger signals like calcium between different receptors (Hwanget al., 2003). Some investigative questions require more than quantitative data, but also thevisual information from imaging. Innovative studies have harnessed spectrofluorometrywith microscopy to investigate cellular signaling with its environment and epithelialcell polarity intact, such as apical and basolateral channels, which contribute to maculadensa calcium signaling (Peti-Peterdi and Bell, 1999). Furthermore, the approach hasbeen applied to characterization of the behavior of cells themselves: quantification ofenzymatic activity has tremendous value in estimating protein content in disease models(Kang et al., 2006b, ADDR). In contrast, imaging-based applications provide the addedbenefits of studying the contribution of cellular polarity and possible interactions of cellswith their environment (Peti-Peterdi et al., 2003). It provides an excellent tool for study ofthe mechanisms, regulation, and functional significance of physiological phenomena likerenin release (Toma et al., 2006). Furthermore, intercellular interactions can be observed.Fluorescence resonance energy transfer (FRET) is a tool based on the energy transferbetween a donor and acceptor pair of fluorophores, which can be used to quantifymolecular dynamics like the interactions between proteins. When the donor and acceptorfluorophores are in close proximity to each other, excitation of the donor results indetectable emission only from the acceptor. The donor-acceptor pair is carefully selectedso that donor emission falls into the specific wavelength for acceptor excitation. Afluorescent donor is excited and its emission energy is quenched through absorption bythe acceptor. Intermolecular FRET from donor to acceptor results only in the detectionof emission from the acceptor. When the donor-acceptor pair is dissociated, FRET canno longer occur (excitation of the donor is no longer quenched by the acceptor), anddonor emission may be detected. Therefore, the efficiency of FRET is determined bythree parameters: distance between the donor and acceptor, overlap between the donoremission spectrum and acceptor absorption spectrum, and the relative orientation of thedonor emission dipole moment to the acceptor absorption dipole moment. FRET canbe quantified by cuvette-based spectrofluorometry experiments or in microscopy imageson a pixel-by-pixel basis. Essentially, the technique capitalizes on the proximity of thefluorescent molecules and can be applied to study protein interactions, conformationalchanges, or enzymatic activity.




A variation on FRET: Renin enzymatic activity assessmentsFluorescence imaging has tremendous potential for the qualitative and quantitative char-acterization of pathophysiological conditions. Although typically used to study proteinstructural interactions, the principles of FRET can be applied to a plethora of other in-vestigations. A recently developed fluorogenic renin substrate (Invitrogen and AnaSpec)makes use of FRET between a donor-acceptor pair linked by a sequence of human an-giotensinogen containing the renin cleavage site at the Leu-Val bond (Wang et al., 1993).In imaging-based applications, the method can be used to visualize the intact intra-renalrenin-angiotensin system, studying the directionality of renin release and activity (Peti-Peterdi et al., 2004). In the absence of active renin enzyme, EDANS fluorescence isquenched by the acceptor molecule DABCYL due to their close proximity and the FRETbetween them. However, when cleaved by renin, the fluorophores dissociate and give riseto bright green EDANS fluorescence. The typical spectrofluorometry reading for a reninactivity assay is shown in Fig. 12.12.2B. This technique allows real-time measurementof renin activity, circumvents the use radioactivity, and is conveniently performed withinminutes, as opposed to conventional renin assays using radioimmuno-methods, whichrequires several days to develop. This novel fluorogenic renin substrate has tremendouspotential to measure renin enzymatic activity in renal cortical tissue homogenates oreven to directly visualize the activity of the intra-renal renin-angiotensin system. BasicProtocol 3 provides quantitative data on the enzymatic activity of renin from kidneyhomogenates in a cuvette-based spectrofluorometer.

In Vitro Tissue Imaging of Renin Release: Isolated Microperfused Tissue, JGA,Renal MedullaMulti-photon fluorescence microscopy provides deep confocal sectioning of living tissuesin detailed subcellular resolution with minimal phototoxicity. This ultimately translatesinto the valuable application of continuous, real-time imaging to the examination ofintegrated, multicellular physiological processes. In combination with the in vitro ex-perimental model, these studies can isolate and examine the effects of a variable ondefined cellular compartments within living specimens. This laser-based technology per-mits three-dimensional imaging, time-lapse studies, and quantitative as well as qualitativeanalysis. In turn, it offers potential applicability to the fields of physiology, pharmacology,anatomy, and pathology within virtually any organ or tissue.

The sensitivity and specificity of multiphoton laser scanning microscopy (MPLSM) makeit ideal for application to the study of subcellular structures within thick tissues and even inthe context of live animals. MPLSM is perfectly suited for optical sectioning up to severalhundred microns deep into living specimens, offering ultra-sensitive and quantitativeimaging of organ functions with a level of temporal-spatial resolution not availablethrough other imaging modalities. For more than a decade, multiphoton microscopyhas been successfully paired with various in vitro and in vivo experimental approachesto study a plethora of different functions across a variety of organ systems, makingit an indispensable tool for research. The dynamics of actin filaments, vesicle release,and the polarity of drug uptake are only some examples of the phenomena that can beinvestigated. The visual data obtained provides an unparalleled insight into the cellularstructurefunction relationships, interactions, feedback loops, and (patho)physiologicalprocesses at play.

The capabilities of MPLSM have been particularly well harnessed in studies of light-scattering tissues such as the kidney. The kidneys are integral to both acute andchronic strategies for blood pressure and volume homeostasis, utilizing hormonal (renin-angiotensin system, RAS) as well as structural (tubuloglomerular feedback, TGF) com-ponents. Using this experimental approach, such integrated processes may be visualized,



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

recorded, and quantified in living tissues or animals at the cellular, or even subcellular,levels. Protein exocytosis, intercellular ionic message transmissions, and fluid transitvelocities may all be captured and measured with this modality. Experimental interven-tions may be used to instigate reactions, and real-time videos have the ability to acquirethe expected results while also uncovering unanticipated reactions to such stimulation.




Quantitative imaging of basic renal functions in health and disease can also providecritical information for characterization of the delivery and effects of therapeutic efforts.

Multi-photon imaging permits sectioning through an entire glomerulus (100 m indiameter), and as such has been used successfully for studies on the isolated microp-erfused afferent arteriole-glomerulus to examine dynamic processes of juxtaglomerularstructures. Figure 12.12.3A shows a representative preparation of a conventional trans-mitted light (DIC) detection image, clearly depicting the entry of the afferent arterioleinto the glomerulus. The use of fluorescent probes permits the detection of cellular andsubcellular structures, and Figure 12.12.3B shows the same preparation with two-photonfluorescence imaging of cellular compartments, like renin granules and plasma mem-branes, in additional detail. With the careful selection of probes, nearly any cellularmicroenvironment can be examined. Acidotropic fluorophores including quinacrine andLysoTracker dyes (Invitrogen) are highly membrane permeant, weakly basic compoundsthat rapidly accumulate in acidic cellular organelles like renin granules. The red dye,R18, stains membranes and can be used to delineate the architecture of vessel walls. Oneof the most commonly used probes, DAPI, is used to define nuclei as shown in Figure12.12.3C.

The minimal cytotoxicity of multi-photon excitation permits continuous imaging of livingtissue specimens, and therefore, real-time imaging of tubuloglomerular feedback (TGF)and renin release mechanisms are possible. Time-lapse imaging allows the study of theeffects of various stimuli on the dynamics of renin release, measured as a reductionof quinacrine fluorescence intensity during granule exocytosis. An image of a typicalpreparation superimposed with the field of interest is illustrated in Figure 12.12.3D. Therelease of renin granular content (loss of green fluorescence intensity) can be quantifiedover the course of the process. Not only degranulation, but also enzymatic activity of thereleased renin (detected as the generation of angiotensin I) can be visualized in real-timeusing a FRET-based renin substrate. Together with imaging the actual renin content,this approach is very useful to monitor the status of the intra-renal renin-angiotensinsystem, an important target of anti-hypertensive therapy. In addition to the ability tostudy integrated processes, the detailed resolution of multiphoton microscopy permitsthe detection of changes in intracellular signaling, as shown by the variation in membranepotentials as assessed by the voltage-sensitive dye, annine-6, in Figure 12.12.3E.

Quantitative Imaging of Kidney Functions In VivoMulti-photon microscopy has driven many recent advances in the knowledge of renal(patho)physiological processes: visualization of cellular variables like cytosolic calciumor pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the jux-taglomerular apparatus (JGA), real-time imaging of tubuloglomerular feedback (TGF)and renin release mechanisms. Structures below the surface of the kidney in the cortex,such as the cortical collecting duct and intracellular vesicles, may be clearly visualized inthe same plane as other structures like the glomerulus or proximal tubule (Fig. 12.12.4A).The capacity to simultaneously visualize the glomerulus and proximal as well as distalsegments of the nephron permits the direct comparison of structurally connected parts ofthe living kidney. In vivo quantitative multi-photon imaging can be applied to measure-ments of many kidney functions, including glomerular filtration and permeability, concen-tration, dilution, and activity of the intra-renal renin-angiotensin system. Measurementsof the single nephron glomerular filtration rate (SNGFR) provide a prime example of thefunctionally coordinated process that are best visualized and quantified by the simultane-ous observation of different structures. An ideal nephron orientation for SNGFR studiesis shown in Figure 12.12.4B, encountered by selection of the appropriate plane of interestby scanning across and z-sectioning the kidney. The acquisition of new visual data has



Figure 12.12.3 Imaging in vitro preparations microdissected from mouse kidney. (A) A transmit-ted light differential interference contrast (DIC) image demonstrating the afferent arteriole (AA) andattached 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) Fluorescenceimage of a glomerulus using the nuclear stain DAPI (blue) and the membrane-stain TMA-DPH tolabel the podocytes found surrounding glomerular endothelial cells. (D) DIC image of a glomeruluswith afferent arteriole (AA) and attached tubule segment (cTAL) for double perfusion studies. Fluo-rescence image with quinacrine-stained renin granules (green) is superimposed. (E) Pseudocolorimage of an in vitro afferent arteriole-glomerulus preparation stained with the Stark-shift voltagesensitive dye, annine-6, showing variations in membrane potential in individual vascular smoothmuscle cells. For a color version of this gure, see http://www.currentprotocols.com.




Figure 12.12.4 In vivo imaging and quantication of renal functional parameters in a Munich-Wistar rat. Quinacrine (green) is used to label acidic compartments (including renin granules) and70-kDa rhodamine (red) marks plasma in the intravascular space. (A) Various cortical segmentsof the nephron may be visualized by z-sectioning down to 200 m below the surface of thekidney. 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 thesubcellular level. (B) The single nephron glomerular ltration rate (SNGFR) may be measuredtaking xy-t video recordings and calculating the ow of Lucifer Yellow, an extracellular uid 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 uorescence in the PT indicating alkalotic conditions(bicarbonate reabsorption). For a color version of this gure, see http://www.currentprotocols.com.

challenged a number of existing paradigms in renal pathophysiology and ultimately hastremendous promise to provide non-invasive diagnostic and therapeutic tools in the clinic.

The Munich-Wistar rat strain is an ideal experimental model for in vivo imaging ofthe JGA and glomerular functions due to its characteristic superficial glomeruli. Briefly,surgery involves cannulation of the left femoral artery to monitor systemic blood pressureand the left femoral vein for fluorescent dye and fluid infusions. Finally, the left kidneyis exteriorized through a small dorsal incision and the animal is placed on the stage ofa Leica inverted microscope with the exposed kidney placed in a coverslip-bottomedheated chamber bathed in normal saline. Images up to 200-m deep below the kidneysurface can be collected in time-series (xyt) with LCS imaging software. The in vivo



multiphoton model may be used to directly observe and quantify various(patho)physiological parameters of the kidney including glomerular filtration rate (GFR)and permeability, blood flow, tubular flow, urinary concentration/dilution, and renincontent. Furthermore, integrated and complex functions like TGF-mediated oscillationsin glomerular filtration and tubular flow may also be captured. Kidney function maybe quantitatively visualized in health or disease, including the streptozotocin (STZ)-induced type I diabetes model. Numerous non-toxic, water-soluble fluorophores can beused to label specific renal structures, listed in Table 12.12.3. The circulating plasma orintra-vascular space may be labeled red with a 70-kDa dextran-rhodamine B conjugate,especially useful for red blood cell velocity recordings. Tubular segments and, morespecifically, the content of individual renin granules, can be visualized using quinacrinein a manner similar to that used in in vitro applications. The extracellular fluid markerLucifer Yellow and the gold-standard GFR marker inulin (FITC-conjugated) can be usedto measure SNGFR. All of these fluorescent probes can be excited using the same, singleexcitation wavelength of 860 nm (Mai-Tai), and the emitted, non-descanned fluorescentlight can be detected by external photomultipliers.

IMPORTANT NOTE: Protocols using live animals must first be reviewed and approvedby an Institutional Animal Care and Use Committee (IACUC) or must conform togovernmental regulations regarding the care and use of laboratory animals.

FLUORESCENCE STUDIES OF CULTURED CELLS: CELL SIGNALINGSTUDIES, PRIMARY CULTURE, OR CELL LINES

BASICPROTOCOL 1

Cuvette-Based Spectrouorometry to Assess Second-Messenger Signalingin Living CellsThe following protocol details a method by which the effect of a stimulus on cellfunction can be investigated by assessing changes in cellular calcium signaling. A cuvette-based approach is described here, which uses the cell type of interest grown on glasscoverslips that is then diagonally inserted into the cuvette. The cuvette is perfused withvarious solutions with the help of a pump/vacuum and polyethylene tubing lines in/outof the cuvette. In this particular system, the emitted fluorescence (fura-2) is detectedby photometry (counts/sec), but the protocol can be easily adapted to direct imagingapproaches using camera or photomultiplier-based fluorescence imaging systems.

NOTE: See Strategic Planning for more detail.

MaterialsCells of interestFura-2 ratiometric calcium imaging dye (Invitrogen)Dimethyl sulfoxide (DMSO)Krebs Ringer-HCO3 solution (see recipe)Experimental solutionPBS (APPENDIX 2A)MgCl2CaCl2Ionomycin (membrane permeabilizer)24 40mm glass coverslipsCuvette-based spectrofluorometer (Quantamaster-8, Photon Technology) with

heated cuvette holder block (37C) and quartz cuvettesPeristaltic pump, vacuum, polyethylene tubing for cuvette superfusion and fluid

exchange




Load dye into cells of interest1. Grow cells of interest to at least 75% confluence on 24 40mm coverslips cut in

half lengthwise, which will fit in a standard quartz cuvette diagonally.

2. Dissolve one vial (50 g) of fura-2 dye in 3 l DMSO and then dilute into 10 ml ofKrebs Ringer-HCO3 solution, for a final concentration of 10 M fura-2.

3. Load cells with fura-2 for 30 min (see Support Protocol 1).4. Wash in 10 ml Krebs Ringer-HCO3 for 20 min at room temperature in the dark to

remove excess dye.

5. Transfer coverslip to cuvette with 3 ml of Krebs Ringer-HCO3 (37C).6. Perfuse the cuvette containing the cells of interest on coverslip with Krebs Ringer-

HCO3 for 100 sec to ensure appropriate baseline counts.The ratio should reach a plateau, indicating cell-bath equilibrium, before proceedingwith the experiment.

7. Switch perfusate to the experimental solution and continue recording the change influorescence until the ratio reaches a plateau.

An increase in the ratio is evidence of an intracellular calcium release.Quantify changes in calcium signalQuantification of the intracellular calcium concentrations requires calibration with thecell membrane permeabilizer, calcium ionophore ionomycin. Fluorescence intensitiesand ratiometric values in the presence and absence of calcium will be used to calculateabsolute intracellular calcium concentrations. The outputs of a calcium signaling studyare shown in Figure 12.12.2A, and serves as a representation of typical graphs fromwhich numerical calculations may be made.

8. Prepare 50 ml each of the calibration solutions as follows:

a. Rmin: PBS containing 10 mM MgCl2 and 2 mM EGTA.b. Rmax: PBS containing 10 mM MgCl2 and 20 mM CaCl2.

9. Load a new coverslip with fura-2 as in steps 1 to 4.

10. Prepare a 5 mM stock solution of ionomycin dissolved in DMSO.

11. Dilute 50 l ionomycin stock into 49.95 ml of Rmin to make a 5 M solution ofionomycin dissolved in Rmin.

12. Perfuse coverslip with 5 M ionomycin/Rmin solution for at least 1000 sec or untilthe ratio plateaus at a minimum.

13. After the Ca2+ ratio has reached a minimal value, switch the perfusate to 5 Mionomycin/Rmax (50 l ionomycin into 49.95 ml Rmax) for 500 sec, or until theratio plateaus.

14. To calculate intracellular calcium concentration changes, use the following formula:

[Ca2+]i = Kd (Sf2/Sb2) [(R Rmin)/(Rmax R)]where, R = ratio obtained from experiment at alternate 340/380 nm excitation and510 nm emission, Kd = 224 nM, Rmin = value of the minimum Ca2+ ratio (inabsence of Ca2+), Rmax = value of the maximum Ca2+ ratio (in presence of Ca2+),Sf2 = value of the counts for 380 nm excitation in the absence of Ca2+, and Sb2 =value of the counts for 380 nm excitation in the presence of Ca2+.



SUPPORTPROTOCOL 1

Enhanced Calcium Loading Techniques with Fura-2 and Fluo-4Calcium dye loading protocols may need to be optimized for different cell types. If flu-orescence intensities are insufficient, dyes may need to be loaded along with reagents toprevent dye extrusion from cells. For example, to measure intracellular calcium concen-trations in vascular smooth muscle cells, cells may be loaded with fura-2 (Invitrogen).Fura-2 is dissolved in a 20% pluronic acid-DMSO solution and diluted in Krebs Ringer-HCO3 solution to reach a final concentration of 3M. The dye is then loaded for 30 min atroom temperature together with 2.5 mM Probenecid, an organic anion transport blockerthat prevents dye leakage. Cells are ready for experiments after loading.

A good alternative to fura-2 is the emission ratiometric dye pair fluo-4/Fura Red. Bothdyes can be excited, e.g., by the argon laser at 488 nm, but emission is detected in twoseparate channels, green (fluo-4, peak at 520 20 nm) and red (Fura Red, >600 nm).The fluorophores are diluted to a final concentration of 1 M and loaded with 250 Msulphinpyrazone to prevent dye leakage for 15 min at room temperature.

BASICPROTOCOL 2

Cuvette-Based Spectrouorometry to Assess Nitric Oxide ProductionThe appropriate selection of dyes permits the application of cuvette-based spectrofluo-rometry to studying a countless variety of second-messenger signals. The Basic Protocoldescribes a method to study intracellular calcium changes, and the following protocoloutlines a method for studying the production of nitric oxide, another important second-messenger. The gas nitric oxide (NO) is a free radical that diffuses across the plasmamembrane to affect nearby cells by activating its target, the enzyme guanylate cyclase,which then produces the second messenger cyclic guanosine monophosphate (cGMP).Alternatively, NO can also covalently modify proteins or their metallic cofactors. NOserves many functions, including the relaxation of blood vessels, the regulation of neuro-transmitter exocytosis, cellular immunity, and the activation of apoptosis. It is producedpredominantly from endothelial cells, neutrophils, and macrophages. The following pro-tocol has been specifically tailored to work with endothelial cells, but may be modifiedfor other cell types.

MaterialsEndothelial cells of interestDAF-FM diacetate (nitric oxide imaging dye; Invitrogen)Dimethyl sulfoxide (DMSO)Krebs Ringer-HCO3 solution (see recipe), 37CSodium nitroprusside (SNP; Sigma)Glass coverslipsCuvette-based spectrofluorometer (Quantamaster-8, Photon Technology) with

heated cuvette holder block (37C) and quartz cuvettesLoad dye1. Grow endothelial cells to at least 75% confluence on long glass coverslips.

2. Prepare DAF-FM dye in the dark. Dissolve 50g DAF-FM diacetate in 10l DMSO,for a 2 M final concentration.

3. Dilute DAF-FM/DMSO into 10 ml of Krebs Ringer-HCO3 solution.

4. Load cells on coverslip with the above 2 M DAF-FM solution for 10 min at roomtemperature in the dark.

5. Wash cells in 10 ml Krebs Ringer-HCO3 for 15 min at room temperature in the dark.

6. Transfer coverslip to cuvette with 3 ml of Krebs Ringer-HCO3 (37C).




Measure NO production7. Perfuse cells with Krebs Ringer-HCO3 solution (control solution) for 100 sec to

ensure appropriate baseline counts.Signal should gradually decay in control solution.

8. Switch perfusate to the experimental solution and continue recording the change influorescence.

An increase in the slope is an indication of the production of NO.Verify NO productionA positive control experiment is performed to ensure cell viability and intact NO synthesismachinery. The cells on coverslip are perfused with the NO donor, SNP, and the DAF-FMsignal should increase as an indication of NO production.

9. Dissolve 2.9995 mg SNP in 10 ml of Krebs Ringer-HCO3 solution.

10. Add 90 ml of Krebs Ringer-HCO3 solution to make 100 ml total of a 100 M SNPsolution.

11. Load a new coverslip with DAF-FM as in steps 1 through 4.

12. Perfuse coverslip with 100 M SNP solution for 500 sec, fluorescence signal shouldbe constantly increasing.

BASICPROTOCOL 3

A NOVEL APPLICATION OF FRET: CUVETTE-BASEDSPECTROFLUOROMETRY TO EVALUATE CELLULAR ENZYMEACTIVITYThe following protocol details a method by which renal tissue renin enzyme activity canbe measured in real-time by using a fluorescent renin substrate. A no-flow cuvette-basedapproach is described here, which uses all elements of the enzymatic reaction addedstep-by-step in the cuvette. In this particular system, the emitted fluorescence (EDANS,fluorogenic renin substrate) is detected by photometry (counts/sec), but the protocol canbe easily adapted to direct imaging approaches using camera or photomultiplier-basedfluorescence imaging systems.

NOTE: See Strategic Planning for more detail.

MaterialsMale 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 shaker1-ml microcentrifuge tubesCuvette-based spectrofluorometer (Quantamaster-8, Photon Technology)

Collect kidney cortical homogenate protein1. Sacrifice male mice (C57Bl/6, 20 g, 6 to 8 weeks old) by Inactin injection

(500 mg/kg b.w. i.p.).2. Remove kidneys and capsule. Slice kidney into small sections and weigh tissue.



3. Add tissue to protease inhibitor diluted in homogenization buffer using the followingparameters:

1 kidney 100 mg = 300 l homogenization buffer and 6 l protease inhibitor4. Homogenize sample for 2 min at maximum speed using a tissue homogenizer.

5. Agitate sample for 2 hr at 4C in a cold room on an orbital shaker at 300 rpm.

6. Transfer lysate to 1-ml microcentrifuge tubes.

7. Centrifuge 20 min at 9300 g, 4C. Collect supernatant.8. Quantify protein content (by Bradford assay) in each sample to ensure equal loading

in enzyme activity assays.

Perform renin activity assay in tissue samples9. Warm renin assay buffer to 37C.

10. Set the spectrofluorometer for assay: excitation at 340 nm, emission at 490 nm.

11. Add 3 ml of renin assay buffer and 6 l of renin-FRET substrate to the cuvette.

12. Start baseline reading for 500 sec, during which the signal should gradually decay.

13. Remove contents of cuvette and repeat step 11.

14. Start reading the fluorescence, pausing after 100 sec to add homogenized tissuesample (normalized to at least 10 g of protein) to the cuvette.

The slope of the fluorescence within the first 50 sec of the addition of the tissue providesan estimate of ANG I production/renin enzymatic activity.

BASICPROTOCOL 4

IN VITRO TISSUE IMAGING OF RENIN RELEASE: ISOLATEDMICROPERFUSED TISSUE, JGA, RENAL MEDULLAThe following procedure involves microdissection of an afferent arteriole-attachedglomerulus preparation from a sacrificed animal. Alternatively, a preparation with acortical thick ascending limb of the loop of Henle (cTAL) and attached glomerulus mayalso be dissected. The preparation is then placed on the microscope so the focal plane ofinterest is in the field of view. Fluorescent dyes are loaded by perfusion, and real-timevideo recordings of any variety of processes, including renin release, may be acquiredby multiphoton microscopy. An inverted microscope is useful if the imaging approach iscombined with micromanipulation of the tissue sample from above (like microperfusionof dissected blood vessels described in this unit). Perfusion systems may be customized tofit the needs of the experiment. Vestavia Scientific provides complete perfusion systemsas well as interchangeable components (manipulator, stage plate, perfusion chamber)that are readily applicable for experiments on tubular structures, like those dissected fromthe kidneys. Major commercial confocal microscope systems include the Leica TCSSP5, Zeiss 510 Meta, Olympus Fluoview 1000, etc. Most microscopes can be poweredby broad-band, femtosecond, fully automated, infrared (tunable between 700 and1040 nm) combined photo-diode pump lasers and mode-locked titanium:sapphire lasers(major brands include the Mai-Tai lasers from Spectra-Physics and the Chameleon fromCoherent) for multiphoton excitation. For conventional, one photon-excitation confocalmicroscopy, a variety of visible and UV lasers are commercially available, e.g., the redHeNe (633 nm/10 mW), orange HeNe (594 nm/2 mW), green HeNe (543 nm/1.2 mW),and blue Ar (458 nm/5 mW; 476 nm/5 mW; 488 nm/20 mW; 514 nm/20 mW) lasers.The following protocol outlines recommendations for detecting renin release from theJGA (via changes in quinacrine fluorescence intensity) when switching from the controlKrebs Ringer-HCO3 solution to a different solution of interest.




MaterialsMice (15 to 20 g)Inactin (see recipe)Dissection medium (see recipe)Bath medium (see recipe)95% O2/5% CO2 sourceControl 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-m o. d.)Imaging software (e. g., Leica LCS)

NOTE: Table 12.12.2 provides a list of fluorophores used to specifically label variousstructures or fields of interest within the afferent arteriole-glomerulus complex. Excita-tion/emission parameters and dye loading recommendations are also included.

Microdissect isolated afferent arteriole-juxtaglomerular apparatus-glomeruluspreparation1. Anesthetize mice (15 to 20 g) with Inactin (100 mg/kg b.w. dissolved in water). Cut

renal artery, remove kidney, and sacrifice animal by Inactin overdose (500 mg/kgb.w. i.p.).

2. Detach renal capsule and store kidney in 5 ml of dissection medium at 4C. Prepareslices of coronal sections of kidney and separate medulla. Keep cortex for micro-dissection.

3. Aerate the perfusate/bath medium (a modified Krebs Ringer-HCO3 solution) with95% O2/5% CO2 for 45 min, and adjust the pH to 7.4.

Table 12.12.2 Fluorescent Probes Commonly Used in In Vitro Experiments

Dyea TargetOne photon/multiphotonexcitation (nm)

Loading

TMA-DPH Cell membrane 375/755 1 mM perfusateR18 Cell membrane 543/800 2 mM perfusateHoechst 33342 Nucleus 380/760 2 mM perfusate/bathQuinacrine Acidic granules (renin) 290/860 5 mM perfusate/bathLyso Tracker Red Acidic granules (renin) 598 5 mM perfusateRenin-FRETsubstrate (EDANS)

Renin enzymatic activity 360/720 2 mM bath

Fluo 4 Intracellular calcium 488/850 10 mM perfusate/bathFura Red Intracellular calcium

( ratio = Ca2+)488/850 10 mM perfusate/bath

DAF-FM Nitric oxide 495 10 mM perfusate/bathBCECF pH ( ratio = pH) 495/440 10 mM perfusate/bathNile Red Neutral lipids 543/800 2 mM bathaTMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate; EDANS, 5-(2-amino-ethylamino) 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).



4. Dissect a superficial afferent arteriole with glomerulus and attached distal tubulecontaining the macula densa.

5. Transfer the preparation to a thermoregulated Lucite chamber mounted onto a Leicainverted microscope.

6. Position the preparation at room temperature so the field of interest is in the optimalfocal plane.

Load dye and prepare for perfusion7. Load the preparation with the selected dye to the control arteriolar (or tubular)

perfusate, according to purposes of the study.The loading conditions must be optimized for each dye, after which they are removedfrom both lumens. Adding the fluorophore through the microperfusate offers the advan-tage of much faster loading compared to incubation with dye in the medium. For example,perfusion loading with the pH dye BCECF requires only 4 to 5 min to attain the samefluorescence signal/intensity as a 30- to 45-min incubation for cell cultures. The followingsteps outline recommendations for detecting changes in quinacrine intensity (renin re-lease) when switching from the control Krebs Ringer-HCO3 solution to a different solutionof interest.

8. Cannulate the afferent arteriole and perfuse with a 35-m o. d. glass pipet. Maintainperfusion pressure at 50 mmHg (1 psi) throughout the experiment.

The preparation may also be microdissected with the distal tubule and macula densa intactfor downstream studies. Cannulate the distal tubule segment, perfusing at a baseline rateof 2 nl/min. Tubulo-glomerular feedback may be activated by increasing the rate oftubular perfusion from baseline at 2 nl/min up to 20 nl/min, using a constant 10 mM NaClin the perfusate.

9. After cannulation, gradually raise the temperature in the bath to 37C for the remain-der of the experiment.

10. Continuously aerate the bath with 95% O2/5% CO2.11. Load the dye through the microperfusate according to the specifications for each

dye, see Table 12.12.2 for recommendations.For example, in the case of the acidic granule dye quinacrine, perfuse a 25 M con-centration for 5 min and then wash out with Krebs Ringer-HCO3 for 30 min to permitstabilization of fluorescent signals.

Image renin release12. Set up the laser settings to the quinacrine excitation wavelength of 860 nm, and

emission is collected at 510 nm.13. Collect images in time-series (xyt) with the imaging software (e. g., Leica LCS).

The rate of image acquisition and the duration of study are at the discretion of theexperiment.

To image renin release, visualized as loss of quinacrine fluorescence intensity, one frameshould be captured every 10 sec for 30 min of perfusion with solution of interest.

14. Switch the perfusion solution to the solution of interest.

15. Collect images in time-series (xyt) for 30 min while perfusing with solution ofinterest.

16. Measure fluorescence intensity (8 or 12 bit), as well as vascular and glomerulardiameter values (can be measured with most imaging software).




BASICPROTOCOL 5

QUANTITATIVE IMAGING OF KIDNEY FUNCTIONS IN VIVO BYMULTIPHOTON EXCITATION LASER SCANNING FLUORESCENCEMICROSCOPYThe methods of red blood cell velocity and single nephron glomerular filtration ratemeasurements are briefly described in this protocol. For the quantitative intravital imagingof other renal functions, see UNIT 12.9. Preparations can be visualized using a two-photonlaser scanning fluorescence microscope, such as a Leica TCS SP2 AOBS MP confocalmicroscope system. The Leica LCS imaging software allows collection of images astime-series videos (xyt) and line-scans (xt), permitting visualization and quantificationof physiological function as previously described.

Three water-soluble fluorophores are used to label specific structures. A 70-kD dextran-rhodamine B conjugate (Invitrogen) labels the plasma red. Tubular segments and reningranules are visualized in green with quinacrine (Sigma). Fluorescent probes are excitedat a wavelength of 860 nm (Mai-Tai) and the emitted fluorescent light is detected bytwo-channel (red and green) external photomultipliers.The following is one example of animal preparation. For alternatives, see the SupportProtocol in UNIT 12.9.

MaterialsC57 BL/6 mice or Munich-Wistar ratsInactin (see recipe)KetamineKrebs 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 CSobjective lens (Leica)

Perform surgery1. Anesthetize C57 BL/6 mice with Inactin (thiobutabarbital) at 50 mg/kg and then

inject with Ketamine at 50 mg/kg.Munich-Wistar rats are anesthetized with Inactin at 120 mg/kg.

2. Cannulate the trachea to facilitate breathing using a piece of 0.86-mm i. d. polyethy-lene tubing.

3. Cannulate the left femoral vein for fluid and dye infusion using a piece of 0.86-mmi. d. polyethylene tubing.

4. Cannulate the left femoral artery for systemic blood pressure measurements using apiece of 0.86-mm i. d. polyethylene tubing and an analog single-channel transducersignal conditioner.

Calibration can be performed using a pressure manometer model no. PM-015 and datacan be collected with the data acquisition system QUAD-161.

5. Make a small left dorsal incision to exteriorize the kidney.

6. Give bolus injections of dyes through the femoral vein, avoiding light, and transferanimal to the microscope.

Typically, a red dye like 70-kDa rhodamine B conjugate is used as a plasma marker andthe green stain quinacrine may be used to label acidic granules. For rhodamine, 50 l of



a 10 mg/ml stock dye is diluted in 50 l Krebs Ringer-HCO3. For quinacrine, 50 l of a25 mg/ml stock dye is diluted in 50 l Krebs Ringer-HCO3. Each 100-l dye is given bybolus injection and washed in with 100 l Krebs Ringer-HCO3.

7. Place the animal on the stage of the inverted microscope, with the kidney in thecoverslip-bottomed heated chamber bathed in modified Krebs Ringer-HCO3 buffer.

Perform real-time in vivo imaging8. Visualize the kidney from below using a HCX PL APO 63/1.4NA oil CS objective

lens.High-resolution images can be acquired 150-m deep below the surface of the renalcortex.

9. Excite the tissue at a wavelength of 860 nm to collect fluorescence emissions at590 nm for the red rhodamine vascular signal and 510 nm for the green quinacrineacidic compartment signal.

10. Move the stage in x, y, or z planes to obtain maximal visual information from thekidney.

Acquire red blood cell velocity measurementsThe 70-kDa rhodamine is used as a plasma marker because it is large enough to stayin the vascular compartment and is not taken up by erythrocytes. Therefore, the transitof dye-excluding red blood cells can be captured and measured in repetitive line-scansthrough a central linear axis of the vessel over time. The motion of red blood cellscorrelates with the dark bands: distance corresponds to the movement of the dark bandacross the entire horizontal x-axis and time corresponds to the vertical y-axis distancetraveled for the red blood cell to cross the entire field. Red blood cell velocity ultimatelyprovides an estimate of renal blood flow and is an important hemodynamic parameter inthe assessment of general renal function.

11. Estimate renal blood flow by measuring RBC velocity in cortical capillaries. Takean xt-scan of RBC motion across a capillary, capturing images with a 1-msec timeresolution.

12. Measure x (distance traveled across the axis) and t (time elapsed for transit, inthe y-plane). Divide the two variables to obtain velocity in mm/sec.

For more technical details, see UNIT 12.9. An example of red blood cell velocity measure-ments, an xt line-scan performed in renal peritubular capillaries can be found in Figure12.9.3B

Acquire single nephron glomerular filtration rateThe single nephron glomerular filtration rate (SNGFR) is one possible means of assessingrenal function. In the same optical plane, a superficial glomerulus with a clear openinginto a proximal tubule of at least 100 m in length must be selected. The SNGFR is calcu-lated by measuring the clearance of the extracellular fluid marker, Lucifer Yellow (LY),whose emission is collected at 528 nm. Using high-temporal resolution, the fluorescenceintensity changes of LY are measured at the opening of proximal tubule and downstreamat least 100 m. The duration of time for peak fluorescence intensity shifts at proximaland distal regions of interest in the proximal tubule corresponds to the SNGFR. LY isgiven by bolus i.v. injection, appears in the glomerulus within 5 sec, and is freely filteredinto Bowmans space and the early proximal tubule.

13. Take a video (xyt scan) of at least a 10-sec duration.14. Give a single i.v. bolus infusion of the fluid marker LY into the femoral vein.

15. Calculate the SNGFR as volume traveled over time.


16. Measure the internal diameter, length of the tubule, and transit time of filtrate betweentwo regions of interest in the proximal tubule using the Quantify package of the Leicaconfocal software.

The midpoint of the dye bolus, approximated by the maximal fluorescence intensity,travels at the same speed as the mean fluid velocity, so the transit time (shift between theintensity plots at the two locations) is calculated at the peaks. By calculating tubular fluidvolume [length (diameter/2)2 ], the absolute value of SNGFR can be calculated(volume/time).

BASICPROTOCOL 6

IN VIVO IMAGING OF CYTOSOLIC PARAMETERS (Ca2+, pH)In in vitro model systems, cell cultures and isolated, microperfused renal tissue techniqueshave been widely used in combination with fluorescence imaging to measure cytosolic ionconcentrations and variables including pH, Ca2+, Na+, Cl, cell volume, etc. However,it may be desirable to confirm if these measurements of cellular processes are relevantto in vivo conditions that also exist in the intact kidney. One- or two-photon excitationconfocal imaging, depending on tissue depth, is ideally suited to achieve this task withclose to real-time, subcellular resolution. Figure 12.12.4C demonstrates that it is possibleto measure cytosolic ion concentrations (e. g., pH) in superficial tubular segments (bothin proximal and distal tubules) with conventional one-photon fluorescence excitation.For optical sectioning of deeper structures, two-photon excitation may be required.Loading of tubular epithelial cells with a fluorophore is technically the easiest in thedistal nephron taking advantage of the high luminal dye concentrations there attainedby the renal concentrating mechanism. Using mice, a single i.v. bolus injection of thepH sensitive dye BCECF injected under the renal capsule (Invitrogen, 50 g AM formdissolved in 1 l DMSO and diluted in 50 l Krebs Ringer-HCO3 solution, excitation at488 nm by the argon laser or at 800 nm by the MP laser, emission at 530 nm) providessufficient labeling of tubular cells. In preliminary assays in mice, loading required 5 to10 min, during which time BCECF fluorescence intensity stabilized at values of at leastone order of magnitude greater than background fluorescence. Figure 12.12.4C showsthat BCECF fluorescence intensity was the highest (indicating high, alkalotic pHi) at

Table 12.12.3 Dyes Used for In Vivo Imaging Studies

Dye Target Laser settings Solution Volume Administration

70-kDadextran-rhodamine B(Invitrogen)

Circulatingplasma/intravascularspace

Ex: 860 nmEm: 590 nm

50 l of a 10 mg/mlstock + 50 lRinger

100 l i.v. bolus

Quinacrine(Sigma)

Acidic granules(renin)


50 l of a 25 mg/mlstock + 50 lRinger

100 l i.v. bolus

Lucifer Yellow(Invitrogen)

Extracellularfluid marker


10 l of a 10 mg/mstock + 90 lRinger

100 l i.v. bolus

BCECF(Invitrogen)

pH Ex: 800 nmEm: 530 nm

50 g dye dissolved in1 l DMSO + 49 lRinger

50 l Injection underrenal capsule

Fluo-4(Invitrogen)

Calcium Ex: 488 nmEm: 520 nm

50 g AM dyedissolved in 1 lDMSO + 49 lRinger

50 l i.v. bolus



the brush-border membrane region, consistent with significant bicarbonate reabsorptioninto the relatively small cytosolic volume of apical microvilli. Developing a reproduciblemethod to measure cytosolic pH in tubular cells in vivo will be an important tool todirectly assess the function and activity of ion transport processes in the nephron and saltand water reabsorption under various conditions.

To execute this procedure, perform all steps of Basic Protocol 5 with the exception ofstep 6, giving only the 70-kDa rhodamine B and particular dye of interest (e. g., BCECFor fluo-4) at the concentrations delineated in Table 12.12.3.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2 A; for suppliers, see SUPPLIERS APPENDIX.Bath medium

Dissolve the following in 100 ml water:1 vial (15.6 g) DMEM/F12 (Sigma)0.12 g NaHCO30.5 g BSAAdjust pH of medium to 7.4 with 1 N NaoH or 1 N HClAerate with 5% CO2 prior to usePrepare fresh

Control tubular perfusateDissolve the following in 1 liter of water:0.5844 g NaCl (10 mM)0.3728 g KCl (5 mM)26.352 g N-methyl-D-glucamine (NMDG)-cyclamate (135 mM)0.1203 g MgSO4 (1 mM)0.2273 g Na2HPO4 (1.6 mM)0.0480 g NaH2PO4 (0.4 mM)6 ml CaCl2 (1.5 mM)0.9008 g D-glucose (5 mM)2.3831 g HEPES (10 mM)Adjust pH to 7.4 with 1 N NaOH or 1 N HClVacuum filter with 0.2-m filter in sterile hoodWarm to 37C and aerate with 5% CO2 with each useStore in sterile conditions up to 1 month at 4C

Dissection mediumDissolve the following in 1 liter of water:1 vial DMEM/F121.2 g NaHCO3Adjust pH to 7.4 with 1 N NaOH or 1 N HClVacuum filter solution with 0.2-m filter in sterile hoodAdd 3% FBS (30 ml)Heat to 37C and aerate with 5% CO2 prior to each useStore up to 1 month at 4C

InactinDissolve 120 mg Inactin in 1 ml water. Make fresh for each use. Inactin is light-

sensitive; keep wrapped in foil until it is used.Inject 0.2 ml Inactin for a 200 g rat.




Krebs Ringer-HCO3Dissolve the following into 1 liter of water:6.7206 g NaCl (115 mM)0.3728 g KCl (5 mM)0.1444 g MgSO4 (1.2 mM)0.0341 g Na2HPO4 (0.24 mM)0.1152 g NaH2PO4 (0.96 mM)0.9909 g D-glucose (5.5 mM)8 ml CaCl2 (2 mM)2.1003 g NaHCO3 (25 mM)10 g BSA0.01742 g L-arginine (0.1 mM)Adjust pH to 7.4 with 1 N NaOH or 1 N HClVacuum filter with 0.2-m filter in a sterile hoodWarm to 37C and aerate with 5% CO2 with each useStore in sterile conditions up to 1 month at 4CAlways incubate to 37C and pH solution to 7.4 prior to each use.

For in vivo infusions, supplement Krebs Ringer-HCO3 with BSA (0.35 g BSA/10 ml KrebsRinger-HCO3).

Renin assay buffer100 mM sodium chloride50 mM Tris baseAdjust pH to 8.0 with 1 N NaOH or 1 N HClStore up to 6 months at 4C

Renin-FRET substrateDissolve 1.0 mg renin substrate (AnaSpec) in 438.5l DMSO. Store up to 3 months

at 4C in the dark.

Tissue homogenization bufferAdd 20 mM TrisCl and 1 mM EGTA. Adjust to pH 7.0 with 1 N NaOH or 1 N HCl.

Store up to 6 months at 4C.

COMMENTARYBackground Information

Over the last two decades, confocal micro-scopy and, more recently, multiphoton micro-scopy, have become fundamental tools forbiologists and life scientists. Confocal laserscanning microscopy is a valuable tool forobtaining high-resolution images and 3-D re-constructions of living biological tissues. Two-photon excitation is founded on the conceptdeveloped by physicist Maria Goeppert-Mayerthat two photons of equal energies can com-bine in a fluorescent molecule to emit a pho-ton of equivalent excitation as that from theabsorption of a single photon of double the en-ergy. This synergistic interaction necessitatesthat the two photons interact nearly simulta-neously with the fluorescent probe nearly toproduce an emission with a quadratic depen-dence on the excitation light intensity rather

than the linear dependence of conventionalfluorescence. The probability of this combina-tory event is extremely low, but improved byincreasing the number of excitation attemptsmade. Multiphoton laser scanning microscopywas invented by Watt W. Webb, WinfriedDenk, and Jim Strickler (Denk et al., 1990).Multiphoton laser scanning microscopy usessolid state lasers, which emit photons in100-fsec pulses at a rapid repetition rate(80 MHz) with longer wavelengths than agas laser. Near-infrared and infrared light areused for excitation, as the longer wavelengthsallow deeper penetration into tissues whileavoiding the damaging effects of conventionalultraviolet or visible illumination on livingsamples. Briefly, the specimen is illuminatedwith a wavelength twice that of the absorp-tion peak of the selected fluorophore and the



combination of two or more photons only oc-curs at the focal plane, so there is less lightscattering resulting in data with a more robustsignal-to-background ratio. Electrons outsideof the focal plane are not sufficiently excitedto fluoresce and cause bleaching, so the pre-cision of image acquisition does not require aconfocal pinhole.

The early years following the commercialavailability of multi-photon microscopy her-alded an era of fascination with in vivo or-gan imaging. However, interest rapidly shiftedfrom the mere acquisition of aesthetic im-ages to harnessing the powers of the technol-ogy for quantitative imaging techniques. Thenext surge of studies aimed to develop newprocedures or to extend existing fluorescenceimaging methods to directly observe and quan-tify basic physiological parameters of the kid-ney including single nephron glomerular filtra-tion rate (SNGFR), glomerular permeability,blood flow, tubular flow, tubular reabsorption,urinary concentration/dilution, renin contentand release, and integrated functions like thetubuloglomerular feedback (TGF)-mediatedoscillations in GFR and tubular flow. Multi-photon techniques have elucidated countlessdynamic processes including glomerular fil-tration, proximal tubule endocytosis, apopto-sis, microvascular function, protein expres-sion, and renal cysts at the subcellular level.Many of these techniques are described inUNIT 12.9.

Two-photon excitation confocal imaging isideally suited to confirming if measurementsof cellular processes are relevant to, and reflec-tive of, conditions in the intact kidney. Confo-cal microscopy also offers the benefits of closeto real-time and subcellular resolution, whichallows for realistic examination of physiolog-ical processes. Cytosolic ion concentrations,like pH and calcium levels, may be measuredin both superficial proximal and distal tubularsegments with conventional one-photon fluo-rescence excitation. Two-photon excitation al-lows optical sectioning of deeper structures.The method has been applied by neurosci-entists to study ion dynamics in brain slices(Yuste and Denk, 1995) and even in live ani-mals (Svoboda et al., 1999). Cancer researchhas utilized the technology for in vivo stud-ies of angiogenesis (McDonald and Choyke,2003) and metastasis (Wang et al., 2002).

In vitro microperfused tissue modelIntravital multi-photon microscopy was

used to visualize fenestrations of the afferentarteriole endothelium in the renin-expressing

segment first described by Rosivall. The workillustrated that bulk fluid flow in the JGA orig-inated from the afferent arteriolar ultrafiltra-tion of plasma into the JGA interstitium aswell as the flow of glomerular filtrate in theBowmans space back into the extraglomeru-lar mesangium. These studies concluded thatsignificant fluid flow exists in the JGA, whichmay facilitate filtration of released renin intothe renal interstitium (endocrine function) andmay also modulate TGF and renin signals inthe JGA (hemodynamic function). These find-ings challenge the existing paradigm of theJGA as a static and isolated microenvironment.

In vivo animal imagingA few aspects of imaging intact organs

in vivo are described here. For more de-tails on intravital microscopy, see UNIT 12.9.In vivo imaging offers virtually noninvasiveinsight into live organisms and helps charac-terize metabolic processes and disease-relatedchanges in the body. Previously describedmethods have established the use of multipho-ton imaging for the quantitative and qualitativeevaluation of various aspects of renal function,including glomerular filtration and tubular re-absorption (Yu et al., 2005). In vivo studiesexamine the tissues of whole, living organismswith all physiological and regulatory compart-ments intact. The approach is thus ideal fortesting drugs on animals, clinical trials, or toassess the functional significance of any in-tervention. Although the experimental modeldoes not isolate potential confounding compo-nents the way in vitro studies do, the approachis often preferable for investigating the over-all effects and relevance of an experimentalvariable on a living subject. Whether the ob-jective of the study is to compare different in-terventions or to gain general knowledge about(patho)physiology, the relevance and influenceof any factor cannot realistically be evaluatedoutside of the systemic context in which thelocal question resides. Especially with studiesof the therapeutic efficacy of drugs, it wouldbe impossible to truly understand the valueof an intervention independent of potentialmetabolic, regulatory, or compensatory feed-back systems that may play a role in the livingorganism.

Multiphoton excitation fluorescence mi-croscopy is an excellent imaging technique fornon-invasive studies of cell, tissue, and organfunctions in both normal and diseased states.It has the potential to visualize the delivery,site-specific actions, and physiological rele-vance of drugs during the development and




evaluation of therapies. This technique permitsthe observation, manipulation, and discoveryof highly complex and integrative questionsabout the mechanisms underlying pathophys-iological processes (Sipos et al., 2007). Quan-titative imaging with multiphoton microscopymay eventually provide a novel non-invasivediagnostic tool for future clinical applications.For example, the heterogeneity and decline ofrenal function could be detected at the ear-liest stages, prior to the onset of measurableblood and urine signals of underlying pathol-ogy (Kang et al., 2006a). These innovativetechnologies provide the most complex, im-mediate, and dynamic portrayal of physiolog-ical function, clearly depicting and analyzingthe components and mechanisms involved innormal physiology and pathophysiology.

The combination of two-photon technolo-gies with fluorescent probes to illuminate spe-cific previously inaccessible tissues, cells, orintracellular compartments can provide phys-iologically relevant spatio-temporal informa-tion (Komlosi et al., 2006). Preserving thestructural architecture and physiological func-tion of intact tissue is fundamental to studiesof the mechanisms behind physiological andpathophysiological processes. In addition toits superlative descriptive power due to highlysensitive imaging of organ function with ex-traordinary spatial and temporal resolution,this also translates into the collection of quan-titative data. Technological advances in micro-scope design, fluorescent dyes, and analyticalsoftware have synergized to allow subcellu-lar resolution and simultaneous quantitation ofmultiple processes. For over a decade, multi-photon microscopy has been successfully usedwith in vitro and in vivo studies to study var-ious functions of different organs, includingthe kidney and lungs (St. Croix et al., 2006).This imaging technology can bring advancesin the knowledge of pathophysiological pro-cesses across multiple organ systems. Thus,multiphoton microscopy is particularly usefulfor in vivo applications where the ability to vi-sualize events in three dimensions with feed-back mechanisms and humoral signals intact isabsolutely essential, as in the brain (Garaschuket al., 2006). New visual and quantitative datamay challenge existing paradigms in patho-physiology (Rosivall et al., 2006) and havethe potential to eventually provide novel non-invasive diagnostic and therapeutic tools forfuture applications. Whether isolating and re-ducing the field of interest to subcellular mech-anisms, or investigating complex coordinatedprocesses and their molecular footprints, mul-

tiphoton imaging offers an unparalleled powerto observe physiological phenomena and con-textualize their significance.

Critical Parameters andTroubleshootingCells

All of the cuvette-based spectrofluorometryinvestigations of second-messenger signalinghave analogous experimental designs in the invitro multiphoton imaging method. The trans-lation of fluorescence readings into absolutenumbers, as in the case of ratiometric calciumor pH studies, requires calibration with cellsfrom the same plate. Renin enzymatic activ-ity experiments can only be fairly comparedif equivalent amounts of kidney tissue areadded, therefore, appropriate protein concen-tration measurements should be performed toensure equivalent loading of protein betweenexperiments. In some cases, dye washout pe-riods may vary depending on cell type and up-take capacities. The first 100 sec of spectroflu-orometry readings should thus confirm appro-priate behavior of cells and dyes in controlsolutions before proceeding with the experi-ment. All experiments involving fluorescencedyes must be performed in the dark when pos-sible, with minimal exposure to light. For bestreproducibility, do not repetitively freeze andthaw dyes, but rather store in aliquots.

TissueTo ensure the best quality tissue that most

closely approximates living conditions, it isimportant to dissect the preparation within thefirst hour of sacrificing the animal and prefer-ably within the first 15 to 30 min if intact vas-culature is necessary. After the first hour, thetissue begins losing its physiological tonicity,making the dissections more difficult to per-form. To preserve structural architecture andmaintain tissue contents, the tissue must beefficiently positioned in the chamber and thesuperfusion begun immediately. The temper-ature of the sample should then be increasedto 37C as soon as possible after positioningto return the tissue to normal physiologicalconditions. After ensuring proper pipet posi-tions, their smooth perfusion should be en-sured because precipitation of solvents on theglass may sometimes clog the lumen.

Before cannulating the structure of inter-est, there must be no air bubbles in the perfu-sion pipet. Once the preparation is perfusing atthe preferred temperature, the glass may moveout from the focal plane, so the tissue mayneed to be repositioned before start running the



experiments. For this reason, acquiring DICimages are important to verify that fluores-cence signal intensity changes are not an arti-fact of focal plane changes.

AnimalsThe animals themselves are often the most

irregular variable in these studies. Anesthetiz-ing the animals sufficiently, but not exces-sively, is important to maintain physiolog-ical function without eliciting confoundingfeedback mechanisms. To that end, properbreathing, blood pressure, and sedation mustbe carefully monitored throughout all parts ofthe procedure. Keep any fluorescent dyes in thedark to avoid bleaching and all infusions mustbe at physiological pH and temperature to en-sure minimal disturbances to homeostatic con-ditions. Scanning across the renal parenchymaoffers many clues to the condition of the ani-mal. For example, a collapsed collecting ductsignifies dehydration and suggests the need forfluid infusion. Technical complications with invivo animal experiments are minimal if bloodpressure is monitored and fluid hydration sta-tus maintained. To avoid unnecessary tissuedamage by the laser, the imageable surfaceshould be scanned across quickly unless a re-gion of interest requires further evaluation.

Fluorescent dyesWhen selecting the right fluorophore for a

particular study, the specificity of the probesis an important issue. The calcium indicatorsfura-2, fluo-4, and Fura Red used in the pro-tocols all have high affinity and specificityfor calcium. However, some calcium indica-tors fluorescence can be quenched by otherdivalent cations like Mg2+ and Mn2+, there-fore, caution must be used. Visible or infraredlight excited dyes (fluo-4, Fura Red) are pre-ferred over UV-excitable indicators (like fura-2). When the use of UV dyes is unavoidable,the fluorescence excitation should be kept toa minimum (low power, short exposure, longexcitation intervals) when working with livingcells and tissue.

Probably the most specific indicator for ni-tric oxide has been DAF-FM, which is ca-pable of detecting NO in low nanomolaramounts. Other widely used NO indicatorsinclude DAF-2 and 2,3-diaminonaphthalene,which can also detect other free radicals andreactive oxygen species (ROS), so they are lessspecific for NO.

For in vivo applications, the fluorophoresmust be non-toxic, water soluble, and specificfor the organ, cell, or molecular target of inter-est. For example, the dextran-rhodamine con-

jugates, quinacrine, and Lucifer Yellow listedin the protocols have been extensively testedand are FDA-approved for human applications(quinacrine).

Anticipated ResultsAll experiments provide the benefits of not

only visual information, but also quantifiabledata. Second-messenger signaling studies canoffer qualitative confirmation about the pres-ence or absence of an effect. Furthermore,some specific probes like fura-2 for calciumand BCECF for pH can be converted into ab-solute numerical values. In vitro studies withthe same probes offer the same information,with the added benefits of visual confirmationof the effects and the opportunity to incorpo-rate the relevance of cell polarity (apical ver-sus basolateral phenomena) as a component ofthe mechanism. Furthermore, in vitro investi-gations permit the examination of multicellu-lar processes and real-time imaging capturesthe time course in which coordinated eventsare occurring. In vivo studies incorporate allof the above parameters along with the abil-ity to contextualize the subcellular phenomenaand physiological interventions with their rel-evance to systemic functional measurements(like blood pressure, blood flow, and GFR).Ultimately, multiphoton imaging offers theunique ability to visualize, quantify, and com-pare the sequence of events driving pathophys-iological processes on subcellular and intercel-lular levels.

Time ConsiderationsCells

Dye loading for cellular studies may takeanywhere between 5 and 30 min, followed bya 20-min wash in Krebs Ringer-HCO3 solu-tion. Spectrofluorometry experiments to as-sess changes in cellular signaling moleculesare typically run for 500 sec, although somestudies may have definitive answers within100 sec.

TissueDye loading for in vitro perfusion studies

is vastly expedited compared to bath loadingfor cells. Dye loading may take between 5 and10 min, followed by a 5-min wash in KrebsRinger-HCO3 solution. Real-time imaging ex-periments to assess changes in fluorescencesignals are typically run for 20 min, acquiringimages every 5 to 10 sec.

AnimalsAnimal surgeries typically take 30 to

45 min for tracheotomy, arterial and venous




cannulations, kidney exteriorization, and dyeinfusion. The imaging portion of studies ismuch more variable in duration. A single redblood cell velocity recording takes

IMAGING From Single Cell to the Whole Organism

Documents

Transcript of IMAGING From Single Cell to the Whole Organism