IMAGING From Single Cell To The Whole Organism

From In Vitro to In Vivo: Imaging from
the Single Cell to the Whole Organism
UNIT 12.12
Jung Julie Kang,1 Ildiko Toma,1 Arnold Sipos,1 and Janos Peti-Peterdi1
1
University of Southern California, Los Angeles, California
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
C 2008 by John Wiley & Sons,
organism. Curr. Protoc. Cytom. 44:12.12.1-12.12.26.
Inc.
Keywords: in vivo imaging r in vitro imaging r multiphoton fluorescence
microscopy r real-time imaging r intravital imaging r laser-scanning microscopy
INTRODUCTION
The application of in vitro fluorescence imaging to cellular studies permits important
discoveries about structure, function, responses to the environment, intracellular signaling pathways, and, indirectly, intercellular relationships. Such studies are particularly
useful in determining the specific mechanisms by which particular cellular components
contribute to larger processes, such as the roles of endothelial nitric oxide production
in vasodilation or the relevance of vascular smooth muscle calcium concentration to
vasoconstriction. Furthermore, both the acute and chronic effects of various stimuli may
be investigated: varying the culturing media or conditions of cells may cause changes in
cell signaling or function, which can be detected with quantitative imaging. Fluorescence
imaging has tremendous applicability to studies of cytosolic changes (e.g., calcium, pH,
cell volume), cellular responses to stimuli (e.g., nitric oxide, prostaglandins), and protein
content/enzyme activity (e.g., renin). Cellular studies permit the determination of the
mechanisms responsible for propagating complex pathways, providing valuable targets
for intervention, especially in disease.
This unit delineates several different protocols for applying fluorescence imaging technology to cellular studies. The superior image quality and resolution permits the visualization
of morphology in living cells, as shown with primary cell cultures of vascular smooth
muscle in Figure 12.12.1 A,B. The acidophilic fluorophore, quinacrine, may be used
to label renin granules in appropriate cells, shown in Figure 12.12.1 C. Lipid vesicles
may be identified with the red stain, Nile Red, shown in Figure 12.12.1D. The judicious
selection of dyes permits simultaneous co-labeling of multiple intracellular structures,
Current Protocols in Cytometry 12.12.1-12.12.26, April 2008

Published online April 2008 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142956.cy1212s44
C 2008 John Wiley & Sons, Inc.
Copyright
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Imaging
12.12.1
Supplement 44
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
Figure 12.12.1 Fluorescence imaging of primary cultures and cell lines to study morphology
and compartments. DIC (A) and uorescence (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). For a color version
of this gure, see http://www.currentprotocols.com.
12.12.2
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Current Protocols in Cytometry
Table 12.12.1 Dyes Used in Cellular Cuvette-Based Spectrouorometry

Studies
Dye
Parameter measured
Excitation
Emission
Renin-FRET
Substrate
Renin enzymatic activity
340 nm
490 nm
Fura-2
Intracellular calcium
( ratio = Ca2+ )
340/380 nm
510 nm
Fluo 4
488 nm
516 nm
DAF-FM
Nitric oxide
495 nm
515 nm
BCECF
pH ( ratio = pH)
500/440 nm
530 nm
and the use of Mito-Tracker Red for mitochondria and quinacrine for acidic granules in
macula densa cells beautifully illustrates the morphology and highlights the specificity
of each of these dyes for their organelles (Fig. 12.12.1E). The imaging system may also
be used to visualize fixed cells for immunocytochemistry experiments. In addition to
structural characterization, the appropriate selection of fluorescent probes permits the
application of cuvette-based spectrofluorometry to investigate direct cause-and-effect
relationships by studying cytosolic signals and second messengers in response to different stimuli. Table 12.12.1 lists various cellular structural and messenger dyes useful
for spectrofluorometry studies. Quantification of cellular enzymatic activity is another
valuable application of this technique. A recently developed fluorogenic peptide based on
fluorescence resonance energy transfer (FRET) permits real-time measurements of renin
activity and can be used to analyze enzymatic activity of tissue samples from healthy and
diseased animals. Imaging studies on cells have limitless applicability to studying direct
and acute effects as well as evaluating changes in chronic disease conditions.
STRATEGIC PLANNING
Cuvette-Based Spectrouorometry to Assess Second-Messenger Signaling
in Living Cells
Signal transduction refers to the process by which a cell receives input and converts
it to another signal, typically involving an ordered sequence of intracellular reactions
carried out by enzymes and linked through second messengers. The association of stimulus with signal transduction pathway, second messenger, and ultimately, end-response,
provides important mechanistic information that can be utilized to promote favorable
and inhibit detrimental processes. Stimuli that initiate a cellular response may be molecular (hormones, cytokines), environmental (extracellular matrix), or physical (light) in
nature. Some cellular responses to extracellular stimulation that depend on signal transduction include metabolism and cell proliferation/death. Therefore, the translation of
external cues into internal messages that elicit specific cellular actions involves signal
transduction. Many diverse disease processes including diabetes, hypertension, autoimmunity, and cancer arise from defects in signal transduction pathways, elucidating the
importance of signal transduction to physiology as well as pathology.
In vitro cell signaling
Fluorescence microscopy has vast applicability to cell signaling studies. Alterations in
pH (Peti-Peterdi et al., 2000), sodium (Peti-Peterdi et al., 2002a), and nitric oxide (Kovacs
et al., 2003) have all been investigated with commercially available probes. Fluorescence
imaging 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 and
then relayed to target molecules in the cytosol and/or nucleus by second messenger
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molecules. In addition to functioning as molecules that translate extracellular cues into

intracellular 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 dyes
for many second messengers exist, so prudent experimental design can provide definitive
confirmation of the effects of hypothesized inputs on cellular behavior. For example,
the technology may be applied to investigating the effects of a proposed substrate on
a G proteincoupled receptormediated intracellular calcium signal. Furthermore, the
effects of receptor inhibition on the second messenger signal may be analyzed with this
approach. Ultimately, the data obtained can elucidate the influence of a stimulus on a
cell by assessing changes in calcium signaling in larger physiological or pathological
processes (Peti-Peterdi et al., 2002a).
Calcium signaling
Since intracellular signal transduction is largely carried out by second messenger
molecules, identification of changes in second messengers provides evidence of a direct effect of stimulus on cell behavior. Calcium is one of the most widely studied second
messengers 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 concentration
is normally maintained at very low levels by sequestration in the smooth endoplasmic
reticulum and mitochondria. Three main signals that promote the activation and release
of calcium are G proteincoupled receptor-regulated pathways, receptor tyrosine kinase
pathways, and ligand or current-gated ion channels. Its release from the endoplasmic
reticulum 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 fluorescent
dye that binds to free intracellular calcium. Its fluorescence is detected at 510 nm in
response to alternate excitation at 340 and 380 nm, with the ratio of the two (340/380) directly correlating to the amount of intracellular calcium. The use of a ratio resolves some
experimental challenges such as dye concentration and background autofluorescence,
making it an ideal choice for quantitative measurements. Fura-2 is thus the preferential dye for ratiometric calcium imaging when the alternation of excitation wavelengths
is more practical than the detection of multiple emission wavelengths. A newer generation of calcium fluorophores includes fluo-4, which provides an efficient, validated
method of imaging intracellular calcium fluxes. Because fluo-4 AM loads faster and
offers greater fluorescence at equivalent concentrations, it is the preferred indicator for
confocal microscopy, flow cytometry, and microplate screening applications. Although
each fluorescent calcium probe has different benefits, their combined use may provide
an extra level of validation of the data obtained. Fluo-4 and Fura Red respond to [Ca2+ ]i
changes with no significant kinetic differences. However, fluo-4 fluorescence increases
with a rise in [Ca2+ ]i , while Fura Red fluorescence decreases. These features make Fluo-4
and Fura Red an excellent dye pair for ratiometric [Ca2+ ]i imaging (Peti-Peterdi, 2006).
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
The application and choice of various fluorophores depends on the fluorescence equipment 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 that
have similar loading characteristics) are almost always recommended, which exclude
the possibility of artifacts associated with dye loading, photobleaching, leakage, cell
volume changes, etc. For the widely used xenon light source-based systems, fura-2 is
an ideal choice since excitation ratiometric approaches can be used. For instruments
powered by laser sources, such as confocal and multi-photon fluorescence microscopes,
emission ratiometric approaches are typically required because of the single, fixed excitation wavelength. For these systems, the fluo-4/Fura Red ratio pair is the preferred
12.12.4
Supplement 44
selection for calcium imaging. Fluo-4 and Fura Red AM forms (cell membrane permeable for loading) are non-fluorescent, as opposed to fura-2 and indo-1, and do not tend
to compartmentalize, factors that would significantly limit the sensitivity of detecting
cytosolic-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 that
the 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 the
choice for high calcium conditions (Kd = 2.3 M).
A Novel Application of FRET: Cuvette-Based Spectrouorometry to Evaluate

Cellular Enzyme Activity
Cuvette-based spectrofluorometry is valuable for investigations that aim to determine
the presence or absence of a direct, acute effect of a given intervention on cellular
function. In certain circumstances, the magnitude of the effect may even be quantified.
Cuvette-based investigations are readily applicable for the study of specific causes and
effects in isolated cells, providing definitive information about external influences or the
cellular machinery involved. For example, the cuvette system has been applied to study
the potential value of purinergic receptormediated calcium signaling as a potential
rescue for epithelial cells in cystic fibrosis (Zsembery et al., 2003). Alternatively, the
experimental approach has been used to assess intact cellular machinery by comparing
changes in second messenger signals like calcium between different receptors (Hwang
et al., 2003). Some investigative questions require more than quantitative data, but also the
visual information from imaging. Innovative studies have harnessed spectrofluorometry
with microscopy to investigate cellular signaling with its environment and epithelial
cell polarity intact, such as apical and basolateral channels, which contribute to macula
densa calcium signaling (Peti-Peterdi and Bell, 1999). Furthermore, the approach has
been applied to characterization of the behavior of cells themselves: quantification of
enzymatic activity has tremendous value in estimating protein content in disease models
(Kang et al., 2006b, ADDR). In contrast, imaging-based applications provide the added
benefits of studying the contribution of cellular polarity and possible interactions of cells
with their environment (Peti-Peterdi et al., 2003). It provides an excellent tool for study of
the mechanisms, regulation, and functional significance of physiological phenomena like
renin release (Toma et al., 2006). Furthermore, intercellular interactions can be observed.
Fluorescence resonance energy transfer (FRET) is a tool based on the energy transfer
between a donor and acceptor pair of fluorophores, which can be used to quantify
molecular dynamics like the interactions between proteins. When the donor and acceptor
fluorophores are in close proximity to each other, excitation of the donor results in
detectable emission only from the acceptor. The donor-acceptor pair is carefully selected
so that donor emission falls into the specific wavelength for acceptor excitation. A
fluorescent donor is excited and its emission energy is quenched through absorption by
the acceptor. Intermolecular FRET from donor to acceptor results only in the detection
of emission from the acceptor. When the donor-acceptor pair is dissociated, FRET can
no longer occur (excitation of the donor is no longer quenched by the acceptor), and
donor emission may be detected. Therefore, the efficiency of FRET is determined by
three parameters: distance between the donor and acceptor, overlap between the donor
emission spectrum and acceptor absorption spectrum, and the relative orientation of the
donor emission dipole moment to the acceptor absorption dipole moment. FRET can
be quantified by cuvette-based spectrofluorometry experiments or in microscopy images
on a pixel-by-pixel basis. Essentially, the technique capitalizes on the proximity of the
fluorescent molecules and can be applied to study protein interactions, conformational
changes, or enzymatic activity.
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A variation on FRET: Renin enzymatic activity assessments

Fluorescence imaging has tremendous potential for the qualitative and quantitative characterization of pathophysiological conditions. Although typically used to study protein
structural interactions, the principles of FRET can be applied to a plethora of other investigations. A recently developed fluorogenic renin substrate (Invitrogen and AnaSpec)
makes use of FRET between a donor-acceptor pair linked by a sequence of human angiotensinogen 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-renal
renin-angiotensin system, studying the directionality of renin release and activity (PetiPeterdi et al., 2004). In the absence of active renin enzyme, EDANS fluorescence is
quenched by the acceptor molecule DABCYL due to their close proximity and the FRET
between them. However, when cleaved by renin, the fluorophores dissociate and give rise
to bright green EDANS fluorescence. The typical spectrofluorometry reading for a renin
activity assay is shown in Fig. 12.12.2B. This technique allows real-time measurement
of renin activity, circumvents the use radioactivity, and is conveniently performed within
minutes, as opposed to conventional renin assays using radioimmuno-methods, which
requires several days to develop. This novel fluorogenic renin substrate has tremendous
potential to measure renin enzymatic activity in renal cortical tissue homogenates or
even to directly visualize the activity of the intra-renal renin-angiotensin system. Basic
Protocol 3 provides quantitative data on the enzymatic activity of renin from kidney
homogenates in a cuvette-based spectrofluorometer.
In Vitro Tissue Imaging of Renin Release: Isolated Microperfused Tissue, JGA,
Renal Medulla
Multi-photon fluorescence microscopy provides deep confocal sectioning of living tissues
in detailed subcellular resolution with minimal phototoxicity. This ultimately translates
into the valuable application of continuous, real-time imaging to the examination of
integrated, multicellular physiological processes. In combination with the in vitro experimental model, these studies can isolate and examine the effects of a variable on
defined cellular compartments within living specimens. This laser-based technology permits three-dimensional imaging, time-lapse studies, and quantitative as well as qualitative
analysis. 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) make
it ideal for application to the study of subcellular structures within thick tissues and even in
the context of live animals. MPLSM is perfectly suited for optical sectioning up to several
hundred microns deep into living specimens, offering ultra-sensitive and quantitative
imaging of organ functions with a level of temporal-spatial resolution not available
through other imaging modalities. For more than a decade, multiphoton microscopy
has been successfully paired with various in vitro and in vivo experimental approaches
to study a plethora of different functions across a variety of organ systems, making
it 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 be
investigated. The visual data obtained provides an unparalleled insight into the cellular
structurefunction relationships, interactions, feedback loops, and (patho)physiological
processes at play.
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
The capabilities of MPLSM have been particularly well harnessed in studies of lightscattering tissues such as the kidney. The kidneys are integral to both acute and
chronic strategies for blood pressure and volume homeostasis, utilizing hormonal (reninangiotensin system, RAS) as well as structural (tubuloglomerular feedback, TGF) components. Using this experimental approach, such integrated processes may be visualized,
12.12.6
Supplement 44
Figure 12.12.2 Spectrouorometry readings for ratiometric calcium signaling (A) and a reninFRET enzymatic activity assay (B). (A) Representative recording and procedure of calibrating
fura-2 uorescence 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 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 transit
velocities may all be captured and measured with this modality. Experimental interventions may be used to instigate reactions, and real-time videos have the ability to acquire
the expected results while also uncovering unanticipated reactions to such stimulation.
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Quantitative imaging of basic renal functions in health and disease can also provide
critical information for characterization of the delivery and effects of therapeutic efforts.
Multi-photon imaging permits sectioning through an entire glomerulus (100 m in
diameter), and as such has been used successfully for studies on the isolated microperfused afferent arteriole-glomerulus to examine dynamic processes of juxtaglomerular
structures. Figure 12.12.3A shows a representative preparation of a conventional transmitted light (DIC) detection image, clearly depicting the entry of the afferent arteriole
into the glomerulus. The use of fluorescent probes permits the detection of cellular and
subcellular structures, and Figure 12.12.3B shows the same preparation with two-photon
fluorescence imaging of cellular compartments, like renin granules and plasma membranes, in additional detail. With the careful selection of probes, nearly any cellular
microenvironment can be examined. Acidotropic fluorophores including quinacrine and
LysoTracker dyes (Invitrogen) are highly membrane permeant, weakly basic compounds
that 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. One
of the most commonly used probes, DAPI, is used to define nuclei as shown in Figure
12.12.3C.
The minimal cytotoxicity of multi-photon excitation permits continuous imaging of living
tissue specimens, and therefore, real-time imaging of tubuloglomerular feedback (TGF)
and renin release mechanisms are possible. Time-lapse imaging allows the study of the
effects of various stimuli on the dynamics of renin release, measured as a reduction
of quinacrine fluorescence intensity during granule exocytosis. An image of a typical
preparation superimposed with the field of interest is illustrated in Figure 12.12.3D. The
release of renin granular content (loss of green fluorescence intensity) can be quantified
over the course of the process. Not only degranulation, but also enzymatic activity of the
released renin (detected as the generation of angiotensin I) can be visualized in real-time
using 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-angiotensin
system, an important target of anti-hypertensive therapy. In addition to the ability to
study integrated processes, the detailed resolution of multiphoton microscopy permits
the detection of changes in intracellular signaling, as shown by the variation in membrane
potentials as assessed by the voltage-sensitive dye, annine-6, in Figure 12.12.3E.
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
Quantitative Imaging of Kidney Functions In Vivo

Multi-photon microscopy has driven many recent advances in the knowledge of renal
(patho)physiological processes: visualization of cellular variables like cytosolic calcium
or pH, cell-to-cell communication and signal propagation, interstitial fluid flow in the juxtaglomerular 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 in
the 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 distal
segments of the nephron permits the direct comparison of structurally connected parts of
the living kidney. In vivo quantitative multi-photon imaging can be applied to measurements of many kidney functions, including glomerular filtration and permeability, concentration, dilution, and activity of the intra-renal renin-angiotensin system. Measurements
of the single nephron glomerular filtration rate (SNGFR) provide a prime example of the
functionally coordinated process that are best visualized and quantified by the simultaneous observation of different structures. An ideal nephron orientation for SNGFR studies
is shown in Figure 12.12.4B, encountered by selection of the appropriate plane of interest
by scanning across and z-sectioning the kidney. The acquisition of new visual data has
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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. For a color version of this gure, see http://www.currentprotocols.com.
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Figure 12.12.4 In vivo imaging and quantication of renal functional parameters in a MunichWistar 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 m 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 ltration rate (SNGFR) may be measured
taking 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 has

tremendous promise to provide non-invasive diagnostic and therapeutic tools in the clinic.
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
The Munich-Wistar rat strain is an ideal experimental model for in vivo imaging of
the JGA and glomerular functions due to its characteristic superficial glomeruli. Briefly,
surgery involves cannulation of the left femoral artery to monitor systemic blood pressure
and the left femoral vein for fluorescent dye and fluid infusions. Finally, the left kidney
is exteriorized through a small dorsal incision and the animal is placed on the stage of
a Leica inverted microscope with the exposed kidney placed in a coverslip-bottomed
heated chamber bathed in normal saline. Images up to 200-m deep below the kidney
surface can be collected in time-series (xyt) with LCS imaging software. The in vivo
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Supplement 44
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 renin
content. Furthermore, integrated and complex functions like TGF-mediated oscillations
in glomerular filtration and tubular flow may also be captured. Kidney function may
be quantitatively visualized in health or disease, including the streptozotocin (STZ)induced type I diabetes model. Numerous non-toxic, water-soluble fluorophores can be
used to label specific renal structures, listed in Table 12.12.3. The circulating plasma or
intra-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, more
specifically, the content of individual renin granules, can be visualized using quinacrine
in a manner similar to that used in in vitro applications. The extracellular fluid marker
Lucifer Yellow and the gold-standard GFR marker inulin (FITC-conjugated) can be used
to measure SNGFR. All of these fluorescent probes can be excited using the same, single
excitation wavelength of 860 nm (Mai-Tai), and the emitted, non-descanned fluorescent
light can be detected by external photomultipliers.
IMPORTANT NOTE: Protocols using live animals must first be reviewed and approved
by an Institutional Animal Care and Use Committee (IACUC) or must conform to
governmental regulations regarding the care and use of laboratory animals.
FLUORESCENCE STUDIES OF CULTURED CELLS: CELL SIGNALING

STUDIES, PRIMARY CULTURE, OR CELL LINES
Cuvette-Based Spectrouorometry to Assess Second-Messenger Signaling
in Living Cells
BASIC
PROTOCOL 1
The following protocol details a method by which the effect of a stimulus on cell
function can be investigated by assessing changes in cellular calcium signaling. A cuvettebased approach is described here, which uses the cell type of interest grown on glass
coverslips that is then diagonally inserted into the cuvette. The cuvette is perfused with
various solutions with the help of a pump/vacuum and polyethylene tubing lines in/out
of the cuvette. In this particular system, the emitted fluorescence (fura-2) is detected
by photometry (counts/sec), but the protocol can be easily adapted to direct imaging
approaches using camera or photomultiplier-based fluorescence imaging systems.
NOTE: See Strategic Planning for more detail.
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 40mm 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
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Load dye into cells of interest

1. 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 of
Krebs 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 (37 C).
6. Perfuse the cuvette containing the cells of interest on coverslip with Krebs RingerHCO3 for 100 sec to ensure appropriate baseline counts.
The ratio should reach a plateau, indicating cell-bath equilibrium, before proceeding
with the experiment.
7. Switch perfusate to the experimental solution and continue recording the change in
fluorescence until the ratio reaches a plateau.
An increase in the ratio is evidence of an intracellular calcium release.
Quantify changes in calcium signal

Quantification of the intracellular calcium concentrations requires calibration with the
cell membrane permeabilizer, calcium ionophore ionomycin. Fluorescence intensities
and ratiometric values in the presence and absence of calcium will be used to calculate
absolute intracellular calcium concentrations. The outputs of a calcium signaling study
are shown in Figure 12.12.2A, and serves as a representation of typical graphs from
which 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 of
ionomycin dissolved in Rmin.
12. Perfuse coverslip with 5 M ionomycin/Rmin solution for at least 1000 sec or until
the ratio plateaus at a minimum.
13. After the Ca2+ ratio has reached a minimal value, switch the perfusate to 5 M
ionomycin/Rmax (50 l ionomycin into 49.95 ml Rmax) for 500 sec, or until the
ratio plateaus.
14. To calculate intracellular calcium concentration changes, use the following formula:
[Ca2+ ]i = Kd (Sf2 /Sb2 ) [(R Rmin)/(Rmax R)]
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
where, R = ratio obtained from experiment at alternate 340/380 nm excitation and

510 nm emission, Kd = 224 nM, Rmin = value of the minimum Ca2+ ratio (in
absence 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+ .
12.12.12
Supplement 44
Enhanced Calcium Loading Techniques with Fura-2 and Fluo-4

Calcium dye loading protocols may need to be optimized for different cell types. If fluorescence intensities are insufficient, dyes may need to be loaded along with reagents to
prevent dye extrusion from cells. For example, to measure intracellular calcium concentrations 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 RingerHCO3 solution to reach a final concentration of 3 M. The dye is then loaded for 30 min at
room temperature together with 2.5 mM Probenecid, an organic anion transport blocker
that prevents dye leakage. Cells are ready for experiments after loading.
SUPPORT
PROTOCOL 1
A good alternative to fura-2 is the emission ratiometric dye pair fluo-4/Fura Red. Both
dyes can be excited, e.g., by the argon laser at 488 nm, but emission is detected in two
separate 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 M
sulphinpyrazone to prevent dye leakage for 15 min at room temperature.
Cuvette-Based Spectrouorometry to Assess Nitric Oxide Production

The appropriate selection of dyes permits the application of cuvette-based spectrofluorometry to studying a countless variety of second-messenger signals. The Basic Protocol
describes a method to study intracellular calcium changes, and the following protocol
outlines a method for studying the production of nitric oxide, another important secondmessenger. The gas nitric oxide (NO) is a free radical that diffuses across the plasma
membrane 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. NO
serves many functions, including the relaxation of blood vessels, the regulation of neurotransmitter exocytosis, cellular immunity, and the activation of apoptosis. It is produced
predominantly from endothelial cells, neutrophils, and macrophages. The following protocol has been specifically tailored to work with endothelial cells, but may be modified
for other cell types.
BASIC
PROTOCOL 2
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
Load dye
1. Grow endothelial cells to at least 75% confluence on long glass coverslips.
2. Prepare DAF-FM dye in the dark. Dissolve 50 g DAF-FM diacetate in 10 l 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 room
temperature 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 (37 C).
Cellular and
Molecular
Imaging
12.12.13
Supplement 44
Measure NO production
7. 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 in
fluorescence.
An increase in the slope is an indication of the production of NO.
Verify NO production
A positive control experiment is performed to ensure cell viability and intact NO synthesis
machinery. The cells on coverslip are perfused with the NO donor, SNP, and the DAF-FM
signal 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 SNP
solution.
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 should
be constantly increasing.
BASIC
PROTOCOL 3
A NOVEL APPLICATION OF FRET: CUVETTE-BASED

SPECTROFLUOROMETRY TO EVALUATE CELLULAR ENZYME
ACTIVITY
The following protocol details a method by which renal tissue renin enzyme activity can
be measured in real-time by using a fluorescent renin substrate. A no-flow cuvette-based
approach is described here, which uses all elements of the enzymatic reaction added
step-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 can
be easily adapted to direct imaging approaches using camera or photomultiplier-based
fluorescence imaging systems.
NOTE: See Strategic Planning for more detail.
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)
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
Collect kidney cortical homogenate protein

1. 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.
12.12.14
Supplement 44
3. Add tissue to protease inhibitor diluted in homogenization buffer using the following
parameters:
1 kidney 100 mg = 300 l homogenization buffer and 6 l protease inhibitor
4. Homogenize sample for 2 min at maximum speed using a tissue homogenizer.
5. Agitate sample for 2 hr at 4 C 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, 4 C. 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 samples

9. Warm renin assay buffer to 37 C.
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 tissue
sample (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 provides
an estimate of ANG I production/renin enzymatic activity.
IN VITRO TISSUE IMAGING OF RENIN RELEASE: ISOLATED

MICROPERFUSED TISSUE, JGA, RENAL MEDULLA
The following procedure involves microdissection of an afferent arteriole-attached
glomerulus preparation from a sacrificed animal. Alternatively, a preparation with a
cortical thick ascending limb of the loop of Henle (cTAL) and attached glomerulus may
also be dissected. The preparation is then placed on the microscope so the focal plane of
interest is in the field of view. Fluorescent dyes are loaded by perfusion, and real-time
video recordings of any variety of processes, including renin release, may be acquired
by multiphoton microscopy. An inverted microscope is useful if the imaging approach is
combined with micromanipulation of the tissue sample from above (like microperfusion
of dissected blood vessels described in this unit). Perfusion systems may be customized to
fit the needs of the experiment. Vestavia Scientific provides complete perfusion systems
as well as interchangeable components (manipulator, stage plate, perfusion chamber)
that are readily applicable for experiments on tubular structures, like those dissected from
the kidneys. Major commercial confocal microscope systems include the Leica TCS
SP5, Zeiss 510 Meta, Olympus Fluoview 1000, etc. Most microscopes can be powered
by broad-band, femtosecond, fully automated, infrared (tunable between 700 and
1040 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 from
Coherent) for multiphoton excitation. For conventional, one photon-excitation confocal
microscopy, a variety of visible and UV lasers are commercially available, e.g., the red
HeNe (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 the
JGA (via changes in quinacrine fluorescence intensity) when switching from the control
Krebs Ringer-HCO3 solution to a different solution of interest.
BASIC
PROTOCOL 4
Cellular and
Molecular
Imaging
12.12.15
Supplement 44
Materials
Mice (15 to 20 g)
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-m 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.
Microdissect isolated afferent arteriole-juxtaglomerular apparatus-glomerulus

preparation
1. 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/kg
b.w. i.p.).
2. Detach renal capsule and store kidney in 5 ml of dissection medium at 4 C. Prepare
slices of coronal sections of kidney and separate medulla. Keep cortex for microdissection.
3. Aerate the perfusate/bath medium (a modified Krebs Ringer-HCO3 solution) with
95% 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
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
Dyea
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
488/850
10 mM perfusate/bath
Fura Red
( ratio = Ca2+ )
488/850
DAF-FM
Nitric oxide
495
BCECF
pH ( ratio = pH)
495/440
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-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).
12.12.16
Supplement 44
4. Dissect a superficial afferent arteriole with glomerulus and attached distal tubule
containing the macula densa.
5. Transfer the preparation to a thermoregulated Lucite chamber mounted onto a Leica
inverted microscope.
6. Position the preparation at room temperature so the field of interest is in the optimal
focal plane.
Load dye and prepare for perfusion

7. 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 removed
from both lumens. Adding the fluorophore through the microperfusate offers the advantage 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 same
fluorescence signal/intensity as a 30- to 45-min incubation for cell cultures. The following
steps outline recommendations for detecting changes in quinacrine intensity (renin release) when switching from the control Krebs Ringer-HCO3 solution to a different solution
of interest.
8. Cannulate the afferent arteriole and perfuse with a 35-m o. d. glass pipet. Maintain
perfusion pressure at 50 mmHg (1 psi) throughout the experiment.
The preparation may also be microdissected with the distal tubule and macula densa intact
for downstream studies. Cannulate the distal tubule segment, perfusing at a baseline rate
of 2 nl/min. Tubulo-glomerular feedback may be activated by increasing the rate of
tubular perfusion from baseline at 2 nl/min up to 20 nl/min, using a constant 10 mM NaCl
in the perfusate.
9. After cannulation, gradually raise the temperature in the bath to 37 C for the remainder 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 concentration for 5 min and then wash out with Krebs Ringer-HCO3 for 30 min to permit
stabilization of fluorescent signals.
Image renin release

12. 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 the
experiment.
To image renin release, visualized as loss of quinacrine fluorescence intensity, one frame
should 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 of
interest.
16. Measure fluorescence intensity (8 or 12 bit), as well as vascular and glomerular
diameter values (can be measured with most imaging software).
Cellular and
Molecular
Imaging
12.12.17
Supplement 44
BASIC
PROTOCOL 5
QUANTITATIVE IMAGING OF KIDNEY FUNCTIONS IN VIVO BY

MULTIPHOTON EXCITATION LASER SCANNING FLUORESCENCE
MICROSCOPY
The methods of red blood cell velocity and single nephron glomerular filtration rate
measurements are briefly described in this protocol. For the quantitative intravital imaging
of other renal functions, see UNIT 12.9. Preparations can be visualized using a two-photon
laser scanning fluorescence microscope, such as a Leica TCS SP2 AOBS MP confocal
microscope system. The Leica LCS imaging software allows collection of images as
time-series videos (xyt) and line-scans (xt), permitting visualization and quantification
of physiological function as previously described.
Three water-soluble fluorophores are used to label specific structures. A 70-kD dextranrhodamine B conjugate (Invitrogen) labels the plasma red. Tubular segments and renin
granules are visualized in green with quinacrine (Sigma). Fluorescent probes are excited
at a wavelength of 860 nm (Mai-Tai) and the emitted fluorescent light is detected by
two-channel (red and green) external photomultipliers.
The following is one example of animal preparation. For alternatives, see the Support
Protocol in UNIT 12.9.
Materials
C57 BL/6 mice or Munich-Wistar rats
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)
Perform surgery
1. 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. polyethylene tubing.
3. Cannulate the left femoral vein for fluid and dye infusion using a piece of 0.86-mm
i. d. polyethylene tubing.
4. Cannulate the left femoral artery for systemic blood pressure measurements using a
piece of 0.86-mm i. d. polyethylene tubing and an analog single-channel transducer
signal conditioner.
Calibration can be performed using a pressure manometer model no. PM-015 and data
can be collected with the data acquisition system QUAD-161.
5. Make a small left dorsal incision to exteriorize the kidney.

From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
12.12.18
Supplement 44
6. Give bolus injections of dyes through the femoral vein, avoiding light, and transfer
animal to the microscope.
Typically, a red dye like 70-kDa rhodamine B conjugate is used as a plasma marker and
the 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 a

25 mg/ml stock dye is diluted in 50 l Krebs Ringer-HCO3 . Each 100-l dye is given by
bolus 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 the
coverslip-bottomed heated chamber bathed in modified Krebs Ringer-HCO3 buffer.
Perform real-time in vivo imaging

8. 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 renal
cortex.
9. Excite the tissue at a wavelength of 860 nm to collect fluorescence emissions at

590 nm for the red rhodamine vascular signal and 510 nm for the green quinacrine
acidic compartment signal.
10. Move the stage in x, y, or z planes to obtain maximal visual information from the
kidney.
Acquire red blood cell velocity measurements

The 70-kDa rhodamine is used as a plasma marker because it is large enough to stay
in the vascular compartment and is not taken up by erythrocytes. Therefore, the transit
of dye-excluding red blood cells can be captured and measured in repetitive line-scans
through a central linear axis of the vessel over time. The motion of red blood cells
correlates with the dark bands: distance corresponds to the movement of the dark band
across the entire horizontal x-axis and time corresponds to the vertical y-axis distance
traveled for the red blood cell to cross the entire field. Red blood cell velocity ultimately
provides an estimate of renal blood flow and is an important hemodynamic parameter in
the assessment of general renal function.
11. Estimate renal blood flow by measuring RBC velocity in cortical capillaries. Take
an xt-scan of RBC motion across a capillary, capturing images with a 1-msec time
resolution.
12. Measure x (distance traveled across the axis) and t (time elapsed for transit, in
the 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 measurements, an xt line-scan performed in renal peritubular capillaries can be found in Figure
12.9.3B
Acquire single nephron glomerular filtration rate

The single nephron glomerular filtration rate (SNGFR) is one possible means of assessing
renal function. In the same optical plane, a superficial glomerulus with a clear opening
into a proximal tubule of at least 100 m in length must be selected. The SNGFR is calculated by measuring the clearance of the extracellular fluid marker, Lucifer Yellow (LY),
whose emission is collected at 528 nm. Using high-temporal resolution, the fluorescence
intensity changes of LY are measured at the opening of proximal tubule and downstream
at least 100 m. The duration of time for peak fluorescence intensity shifts at proximal
and distal regions of interest in the proximal tubule corresponds to the SNGFR. LY is
given by bolus i.v. injection, appears in the glomerulus within 5 sec, and is freely filtered
into 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.
Cellular and
Molecular
Imaging
12.12.19
Supplement 44
16. Measure the internal diameter, length of the tubule, and transit time of filtrate between
two regions of interest in the proximal tubule using the Quantify package of the Leica
confocal 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 the
intensity plots at the two locations) is calculated at the peaks. By calculating tubular fluid
volume [length (diameter/2)2 ], the absolute value of SNGFR can be calculated
(volume/time).
BASIC
PROTOCOL 6
IN VIVO IMAGING OF CYTOSOLIC PARAMETERS (Ca2+ , pH)

In in vitro model systems, cell cultures and isolated, microperfused renal tissue techniques
have been widely used in combination with fluorescence imaging to measure cytosolic ion
concentrations and variables including pH, Ca2+ , Na+ , Cl , cell volume, etc. However,
it may be desirable to confirm if these measurements of cellular processes are relevant
to in vivo conditions that also exist in the intact kidney. One- or two-photon excitation
confocal imaging, depending on tissue depth, is ideally suited to achieve this task with
close to real-time, subcellular resolution. Figure 12.12.4C demonstrates that it is possible
to measure cytosolic ion concentrations (e. g., pH) in superficial tubular segments (both
in 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 the
distal nephron taking advantage of the high luminal dye concentrations there attained
by the renal concentrating mechanism. Using mice, a single i.v. bolus injection of the
pH sensitive dye BCECF injected under the renal capsule (Invitrogen, 50 g AM form
dissolved in 1 l DMSO and diluted in 50 l Krebs Ringer-HCO3 solution, excitation at
488 nm by the argon laser or at 800 nm by the MP laser, emission at 530 nm) provides
sufficient labeling of tubular cells. In preliminary assays in mice, loading required 5 to
10 min, during which time BCECF fluorescence intensity stabilized at values of at least
one order of magnitude greater than background fluorescence. Figure 12.12.4C shows
that 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-kDa
dextran-rhodamine B
(Invitrogen)
Circulating
plasma/
intravascular
space
Ex: 860 nm
Em: 590 nm
50 l of a 10 mg/ml
stock + 50 l
Ringer
100 l
i.v. bolus
Quinacrine
(Sigma)
Acidic granules Ex: 860 nm

(renin)
Em: 510 nm
50 l of a 25 mg/ml
stock + 50 l
Ringer
100 l
i.v. bolus
Lucifer Yellow
(Invitrogen)
Extracellular
fluid marker
Ex: 860 nm
Em: 528 nm
10 l of a 10 mg/m
stock + 90 l
Ringer
100 l
i.v. bolus
BCECF
(Invitrogen)
pH
Ex: 800 nm
Em: 530 nm
50 g dye dissolved in
1 l DMSO + 49 l
Ringer
50 l
Injection under
renal capsule
Fluo-4
(Invitrogen)
Calcium
Ex: 488 nm
Em: 520 nm
50 g AM dye
dissolved in 1 l
DMSO + 49 l
Ringer
50 l
i.v. bolus
12.12.20
Supplement 44
the brush-border membrane region, consistent with significant bicarbonate reabsorption

into the relatively small cytosolic volume of apical microvilli. Developing a reproducible
method to measure cytosolic pH in tubular cells in vivo will be an important tool to
directly assess the function and activity of ion transport processes in the nephron and salt
and water reabsorption under various conditions.
To execute this procedure, perform all steps of Basic Protocol 5 with the exception of
step 6, giving only the 70-kDa rhodamine B and particular dye of interest (e. g., BCECF
or fluo-4) at the concentrations delineated in Table 12.12.3.
REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 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 NaHCO3
0.5 g BSA
Adjust pH of medium to 7.4 with 1 N NaoH or 1 N HCl
Aerate with 5% CO2 prior to use
Prepare fresh
Control tubular perfusate
Dissolve 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 Na2 HPO4 (1.6 mM)
0.0480 g NaH2 PO4 (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 HCl
Vacuum filter with 0.2-m filter in sterile hood
Warm to 37 C and aerate with 5% CO2 with each use
Store in sterile conditions up to 1 month at 4 C
Dissection medium
Dissolve the following in 1 liter of water:
1 vial DMEM/F12
1.2 g NaHCO3
Vacuum filter solution with 0.2-m filter in sterile hood
Add 3% FBS (30 ml)
Heat to 37 C and aerate with 5% CO2 prior to each use
Store up to 1 month at 4 C
Inactin
Dissolve 120 mg Inactin in 1 ml water. Make fresh for each use. Inactin is lightsensitive; keep wrapped in foil until it is used.
Inject 0.2 ml Inactin for a 200 g rat.
Cellular and
Molecular
Imaging
12.12.21
Supplement 44
Krebs Ringer-HCO3
Dissolve 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 Na2 HPO4 (0.24 mM)
0.1152 g NaH2 PO4 (0.96 mM)
0.9909 g D-glucose (5.5 mM)
8 ml CaCl2 (2 mM)
2.1003 g NaHCO3 (25 mM)
10 g BSA
0.01742 g L-arginine (0.1 mM)
Vacuum filter with 0.2-m filter in a sterile hood
Warm to 37 C and aerate with 5% CO2 with each use
Store in sterile conditions up to 1 month at 4 C
Always incubate to 37 C 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 Krebs
Ringer-HCO3 ).
Renin assay buffer

100 mM sodium chloride
50 mM Tris base
Store up to 6 months at 4 C
Renin-FRET substrate
Dissolve 1.0 mg renin substrate (AnaSpec) in 438.5 l DMSO. Store up to 3 months
at 4 C in the dark.
Tissue homogenization buffer
Add 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 4 C.
COMMENTARY
Background Information
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
Over the last two decades, confocal microscopy and, more recently, multiphoton microscopy, have become fundamental tools for
biologists and life scientists. Confocal laser
scanning microscopy is a valuable tool for
obtaining high-resolution images and 3-D reconstructions of living biological tissues. Twophoton excitation is founded on the concept
developed by physicist Maria Goeppert-Mayer
that two photons of equal energies can combine in a fluorescent molecule to emit a photon of equivalent excitation as that from the
absorption of a single photon of double the energy. This synergistic interaction necessitates
that the two photons interact nearly simultaneously with the fluorescent probe nearly to
produce an emission with a quadratic dependence on the excitation light intensity rather
than the linear dependence of conventional

fluorescence. The probability of this combinatory event is extremely low, but improved by
increasing the number of excitation attempts
made. Multiphoton laser scanning microscopy
was invented by Watt W. Webb, Winfried
Denk, and Jim Strickler (Denk et al., 1990).
Multiphoton laser scanning microscopy uses
solid state lasers, which emit photons in
100-fsec pulses at a rapid repetition rate
(80 MHz) with longer wavelengths than a
gas laser. Near-infrared and infrared light are
used for excitation, as the longer wavelengths
allow deeper penetration into tissues while
avoiding the damaging effects of conventional
ultraviolet or visible illumination on living
samples. Briefly, the specimen is illuminated
with a wavelength twice that of the absorption peak of the selected fluorophore and the
12.12.22
Supplement 44
combination of two or more photons only occurs at the focal plane, so there is less light
scattering resulting in data with a more robust
signal-to-background ratio. Electrons outside
of the focal plane are not sufficiently excited
to fluoresce and cause bleaching, so the precision of image acquisition does not require a
confocal pinhole.
The early years following the commercial
availability of multi-photon microscopy heralded an era of fascination with in vivo organ imaging. However, interest rapidly shifted
from the mere acquisition of aesthetic images to harnessing the powers of the technology for quantitative imaging techniques. The
next surge of studies aimed to develop new
procedures or to extend existing fluorescence
imaging methods to directly observe and quantify basic physiological parameters of the kidney including single nephron glomerular filtration rate (SNGFR), glomerular permeability,
blood flow, tubular flow, tubular reabsorption,
urinary concentration/dilution, renin content
and release, and integrated functions like the
tubuloglomerular feedback (TGF)-mediated
oscillations in GFR and tubular flow. Multiphoton techniques have elucidated countless
dynamic processes including glomerular filtration, proximal tubule endocytosis, apoptosis, microvascular function, protein expression, and renal cysts at the subcellular level.
Many of these techniques are described in
UNIT 12.9.
Two-photon excitation confocal imaging is
ideally suited to confirming if measurements
of cellular processes are relevant to, and reflective of, conditions in the intact kidney. Confocal microscopy also offers the benefits of close
to real-time and subcellular resolution, which
allows for realistic examination of physiological processes. Cytosolic ion concentrations,
like pH and calcium levels, may be measured
in both superficial proximal and distal tubular
segments with conventional one-photon fluorescence excitation. Two-photon excitation allows optical sectioning of deeper structures.
The method has been applied by neuroscientists to study ion dynamics in brain slices
(Yuste and Denk, 1995) and even in live animals (Svoboda et al., 1999). Cancer research
has utilized the technology for in vivo studies of angiogenesis (McDonald and Choyke,
2003) and metastasis (Wang et al., 2002).
In vitro microperfused tissue model
Intravital multi-photon microscopy was
used to visualize fenestrations of the afferent
arteriole endothelium in the renin-expressing
segment first described by Rosivall. The work

illustrated that bulk fluid flow in the JGA originated from the afferent arteriolar ultrafiltration of plasma into the JGA interstitium as
well as the flow of glomerular filtrate in the
Bowmans space back into the extraglomerular mesangium. These studies concluded that
significant fluid flow exists in the JGA, which
may facilitate filtration of released renin into
the renal interstitium (endocrine function) and
may also modulate TGF and renin signals in
the JGA (hemodynamic function). These findings challenge the existing paradigm of the
JGA as a static and isolated microenvironment.
In vivo animal imaging
A few aspects of imaging intact organs
in vivo are described here. For more details on intravital microscopy, see UNIT 12.9.
In vivo imaging offers virtually noninvasive
insight into live organisms and helps characterize metabolic processes and disease-related
changes in the body. Previously described
methods have established the use of multiphoton imaging for the quantitative and qualitative
evaluation of various aspects of renal function,
including glomerular filtration and tubular reabsorption (Yu et al., 2005). In vivo studies
examine the tissues of whole, living organisms
with all physiological and regulatory compartments intact. The approach is thus ideal for
testing drugs on animals, clinical trials, or to
assess the functional significance of any intervention. Although the experimental model
does not isolate potential confounding components the way in vitro studies do, the approach
is often preferable for investigating the overall effects and relevance of an experimental
variable on a living subject. Whether the objective of the study is to compare different interventions or to gain general knowledge about
(patho)physiology, the relevance and influence
of any factor cannot realistically be evaluated
outside of the systemic context in which the
local question resides. Especially with studies
of the therapeutic efficacy of drugs, it would
be impossible to truly understand the value
of an intervention independent of potential
metabolic, regulatory, or compensatory feedback systems that may play a role in the living
organism.
Multiphoton excitation fluorescence microscopy is an excellent imaging technique for
non-invasive studies of cell, tissue, and organ
functions in both normal and diseased states.
It has the potential to visualize the delivery,
site-specific actions, and physiological relevance of drugs during the development and
Cellular and
Molecular
Imaging
12.12.23
Supplement 44
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
evaluation of therapies. This technique permits

the observation, manipulation, and discovery
of highly complex and integrative questions
about the mechanisms underlying pathophysiological processes (Sipos et al., 2007). Quantitative imaging with multiphoton microscopy
may eventually provide a novel non-invasive
diagnostic tool for future clinical applications.
For example, the heterogeneity and decline of
renal function could be detected at the earliest stages, prior to the onset of measurable
blood and urine signals of underlying pathology (Kang et al., 2006a). These innovative
technologies provide the most complex, immediate, and dynamic portrayal of physiological function, clearly depicting and analyzing
the components and mechanisms involved in
normal physiology and pathophysiology.
The combination of two-photon technologies with fluorescent probes to illuminate specific previously inaccessible tissues, cells, or
intracellular compartments can provide physiologically relevant spatio-temporal information (Komlosi et al., 2006). Preserving the
structural architecture and physiological function of intact tissue is fundamental to studies
of the mechanisms behind physiological and
pathophysiological processes. In addition to
its superlative descriptive power due to highly
sensitive imaging of organ function with extraordinary spatial and temporal resolution,
this also translates into the collection of quantitative data. Technological advances in microscope design, fluorescent dyes, and analytical
software have synergized to allow subcellular resolution and simultaneous quantitation of
multiple processes. For over a decade, multiphoton microscopy has been successfully used
with in vitro and in vivo studies to study various functions of different organs, including
the kidney and lungs (St. Croix et al., 2006).
This imaging technology can bring advances
in the knowledge of pathophysiological processes across multiple organ systems. Thus,
multiphoton microscopy is particularly useful
for in vivo applications where the ability to visualize events in three dimensions with feedback mechanisms and humoral signals intact is
absolutely essential, as in the brain (Garaschuk
et al., 2006). New visual and quantitative data
may challenge existing paradigms in pathophysiology (Rosivall et al., 2006) and have
the potential to eventually provide novel noninvasive diagnostic and therapeutic tools for
future applications. Whether isolating and reducing the field of interest to subcellular mechanisms, or investigating complex coordinated
processes and their molecular footprints, mul-
tiphoton imaging offers an unparalleled power

to observe physiological phenomena and contextualize their significance.
Critical Parameters and

Troubleshooting
Cells
All of the cuvette-based spectrofluorometry
investigations of second-messenger signaling
have analogous experimental designs in the in
vitro multiphoton imaging method. The translation of fluorescence readings into absolute
numbers, as in the case of ratiometric calcium
or pH studies, requires calibration with cells
from the same plate. Renin enzymatic activity experiments can only be fairly compared
if equivalent amounts of kidney tissue are
added, therefore, appropriate protein concentration measurements should be performed to
ensure equivalent loading of protein between
experiments. In some cases, dye washout periods may vary depending on cell type and uptake capacities. The first 100 sec of spectrofluorometry readings should thus confirm appropriate behavior of cells and dyes in control
solutions before proceeding with the experiment. All experiments involving fluorescence
dyes must be performed in the dark when possible, with minimal exposure to light. For best
reproducibility, do not repetitively freeze and
thaw dyes, but rather store in aliquots.
Tissue
To ensure the best quality tissue that most
closely approximates living conditions, it is
important to dissect the preparation within the
first hour of sacrificing the animal and preferably within the first 15 to 30 min if intact vasculature is necessary. After the first hour, the
tissue begins losing its physiological tonicity,
making the dissections more difficult to perform. To preserve structural architecture and
maintain tissue contents, the tissue must be
efficiently positioned in the chamber and the
superfusion begun immediately. The temperature of the sample should then be increased
to 37 C as soon as possible after positioning
to return the tissue to normal physiological
conditions. After ensuring proper pipet positions, their smooth perfusion should be ensured because precipitation of solvents on the
glass may sometimes clog the lumen.
Before cannulating the structure of interest, there must be no air bubbles in the perfusion pipet. Once the preparation is perfusing at
the preferred temperature, the glass may move
out from the focal plane, so the tissue may
need to be repositioned before start running the
12.12.24
Supplement 44
experiments. For this reason, acquiring DIC

images are important to verify that fluorescence signal intensity changes are not an artifact of focal plane changes.
jugates, quinacrine, and Lucifer Yellow listed

in the protocols have been extensively tested
and are FDA-approved for human applications
(quinacrine).
Animals
The animals themselves are often the most
irregular variable in these studies. Anesthetizing the animals sufficiently, but not excessively, is important to maintain physiological function without eliciting confounding
feedback mechanisms. To that end, proper
breathing, blood pressure, and sedation must
be carefully monitored throughout all parts of
the procedure. Keep any fluorescent dyes in the
dark to avoid bleaching and all infusions must
be at physiological pH and temperature to ensure minimal disturbances to homeostatic conditions. Scanning across the renal parenchyma
offers many clues to the condition of the animal. For example, a collapsed collecting duct
signifies dehydration and suggests the need for
fluid infusion. Technical complications with in
vivo animal experiments are minimal if blood
pressure is monitored and fluid hydration status maintained. To avoid unnecessary tissue
damage by the laser, the imageable surface
should be scanned across quickly unless a region of interest requires further evaluation.
Anticipated Results
Fluorescent dyes
When selecting the right fluorophore for a
particular study, the specificity of the probes
is an important issue. The calcium indicators
fura-2, fluo-4, and Fura Red used in the protocols all have high affinity and specificity
for calcium. However, some calcium indicators fluorescence can be quenched by other
divalent cations like Mg2+ and Mn2+ , therefore, caution must be used. Visible or infrared
light excited dyes (fluo-4, Fura Red) are preferred over UV-excitable indicators (like fura2). When the use of UV dyes is unavoidable,
the fluorescence excitation should be kept to
a minimum (low power, short exposure, long
excitation intervals) when working with living
cells and tissue.
Probably the most specific indicator for nitric oxide has been DAF-FM, which is capable of detecting NO in low nanomolar
amounts. Other widely used NO indicators
include DAF-2 and 2,3-diaminonaphthalene,
which can also detect other free radicals and
reactive oxygen species (ROS), so they are less
specific for NO.
For in vivo applications, the fluorophores
must be non-toxic, water soluble, and specific
for the organ, cell, or molecular target of interest. For example, the dextran-rhodamine conCurrent Protocols in Cytometry
All experiments provide the benefits of not

only visual information, but also quantifiable
data. Second-messenger signaling studies can
offer qualitative confirmation about the presence or absence of an effect. Furthermore,
some specific probes like fura-2 for calcium
and BCECF for pH can be converted into absolute numerical values. In vitro studies with
the same probes offer the same information,
with the added benefits of visual confirmation
of the effects and the opportunity to incorporate the relevance of cell polarity (apical versus basolateral phenomena) as a component of
the mechanism. Furthermore, in vitro investigations permit the examination of multicellular processes and real-time imaging captures
the time course in which coordinated events
are occurring. In vivo studies incorporate all
of the above parameters along with the ability to contextualize the subcellular phenomena
and physiological interventions with their relevance to systemic functional measurements
(like blood pressure, blood flow, and GFR).
Ultimately, multiphoton imaging offers the
unique ability to visualize, quantify, and compare the sequence of events driving pathophysiological processes on subcellular and intercellular levels.
Time Considerations
Cells
Dye loading for cellular studies may take
anywhere between 5 and 30 min, followed by
a 20-min wash in Krebs Ringer-HCO3 solution. Spectrofluorometry experiments to assess changes in cellular signaling molecules
are typically run for 500 sec, although some
studies may have definitive answers within
100 sec.
Tissue
Dye loading for in vitro perfusion studies
is vastly expedited compared to bath loading
for cells. Dye loading may take between 5 and
10 min, followed by a 5-min wash in Krebs
Ringer-HCO3 solution. Real-time imaging experiments to assess changes in fluorescence
signals are typically run for 20 min, acquiring
images every 5 to 10 sec.
Animals
Animal surgeries typically take 30 to
45 min for tracheotomy, arterial and venous
Cellular and
Molecular
Imaging
12.12.25
Supplement 44
cannulations, kidney exteriorization, and dye

infusion. The imaging portion of studies is
much more variable in duration. A single red
blood cell velocity recording takes <5 sec,
a single SNGFR recording requires <30 sec,
but changes in tubular concentrating/diluting
function may be assessed over 20 min. Multiphoton imaging is minimally invasive and
not destructive to the animal, so imaging may
proceed for several hours for maximal data
acquisition. Animals may then be terminated
or transferred for rescue surgeries.
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1990. Two-photon laser scanning fluorescence
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Garaschuk, O., Milos, R.I., and Konnerth, A. 2006.
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Hwang, E. F., Williams, I., Kovacs, G., Peti-Peterdi,
J., Siroky, B., Rice, W. C., Bates, E., Schweibert,
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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
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Kang, J. J., Toma, I., Sipos, A., McCulloch, F.,
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Peti-Peterdi, J., Fintha, A., Fuson, A. L., Tousson,
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Rosivall, L., Mirzahosseini, S., Toma, I., Sipos, A.,
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Sipos, A., Toma, I., Kang, J. J., Rosivall, L., and
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St. Croix, C.M., Leelavanichkul, K., and Watkins,
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D. W. 1999. Spread of dendritic excitation in
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in vivo. Nat. Neurosci. 2:65-73.
Toma, I., Kang, J. J., and Peti-Peterdi, J. 2006.
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Unraveling the relationship between macula
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Oleynikov, Y., Huttelmaier, S., Zavadil, J.,
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From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
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12.12.26
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IMAGING From Single Cell To The Whole Organism

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IMAGING From Single Cell To The Whole Organism

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From In Vitro to In Vivo: Imaging from

the Single Cell to the Whole Organism

University of Southern California, Los Angeles, California

Current Protocols in Cytometry 12.12.1-12.12.26, April 2008

Current Protocols in Cytometry

Table 12.12.1 Dyes Used in Cellular Cuvette-Based Spectrouorometry

Renin enzymatic activity

molecules. In addition to functioning as molecules that translate extracellular cues into

Current Protocols in Cytometry

A Novel Application of FRET: Cuvette-Based Spectrouorometry to Evaluate

A variation on FRET: Renin enzymatic activity assessments

Current Protocols in Cytometry

Quantitative Imaging of Kidney Functions In Vivo

Current Protocols in Cytometry

challenged a number of existing paradigms in renal pathophysiology and ultimately has

Current Protocols in Cytometry

multiphoton model may be used to directly observe and quantify various

FLUORESCENCE STUDIES OF CULTURED CELLS: CELL SIGNALING

Load dye into cells of interest

Quantify changes in calcium signal

where, R = ratio obtained from experiment at alternate 340/380 nm excitation and

Current Protocols in Cytometry

Enhanced Calcium Loading Techniques with Fura-2 and Fluo-4

Cuvette-Based Spectrouorometry to Assess Nitric Oxide Production

A NOVEL APPLICATION OF FRET: CUVETTE-BASED

Collect kidney cortical homogenate protein

Current Protocols in Cytometry

Perform renin activity assay in tissue samples

IN VITRO TISSUE IMAGING OF RENIN RELEASE: ISOLATED

Microdissect isolated afferent arteriole-juxtaglomerular apparatus-glomerulus

Acidic granules (renin)

Lyso Tracker Red

Acidic granules (renin)

Renin enzymatic activity

a TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate; EDANS, 5-(2-amino-

Current Protocols in Cytometry

Load dye and prepare for perfusion

Image renin release

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

QUANTITATIVE IMAGING OF KIDNEY FUNCTIONS IN VIVO BY

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

a 10 mg/ml stock dye is diluted in 50 l Krebs Ringer-HCO3 . For quinacrine, 50 l of a

Perform real-time in vivo imaging

9. Excite the tissue at a wavelength of 860 nm to collect fluorescence emissions at

Acquire red blood cell velocity measurements

Acquire single nephron glomerular filtration rate

IN VIVO IMAGING OF CYTOSOLIC PARAMETERS (Ca2+ , pH)

Table 12.12.3 Dyes Used for In Vivo Imaging Studies

Acidic granules Ex: 860 nm

Current Protocols in Cytometry

the brush-border membrane region, consistent with significant bicarbonate reabsorption

REAGENTS AND SOLUTIONS

Renin assay buffer

than the linear dependence of conventional

Current Protocols in Cytometry

segment first described by Rosivall. The work

evaluation of therapies. This technique permits

tiphoton imaging offers an unparalleled power

Critical Parameters and

Current Protocols in Cytometry

experiments. For this reason, acquiring DIC

jugates, quinacrine, and Lucifer Yellow listed

All experiments provide the benefits of not