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UNIT 12.12
Jung Julie Kang,1 Ildiko Toma,1 Arnold Sipos,1 and Janos Peti-Peterdi1
1
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,
Cellular and
Molecular
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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.
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Dye
Parameter measured
Excitation
Emission
Renin-FRET
Substrate
340 nm
490 nm
Fura-2
Intracellular calcium
( ratio = Ca2+ )
340/380 nm
510 nm
Fluo 4
Intracellular calcium
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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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).
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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
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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.
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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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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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.
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
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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.
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).
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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
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
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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.
BASIC
PROTOCOL 4
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Materials
Mice (15 to 20 g)
Inactin (see recipe)
Dissection medium (see recipe)
Bath medium (see recipe)
95% O2 /5% CO2 source
Control tubular perfusate (see recipe)
Krebs Ringer-HCO3 solution (see recipe)
Fluorescent dyes of interest (e. g., quinacrine)
Thermoregulated Lucite chamber (Vestavia Scientific)
Confocal microscope system (e. g., Leica TCS SP2, Leica Microsystem)
Glass pipets (35-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.
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
290/860
5 mM perfusate/bath
598
5 mM perfusate
Renin-FRET
substrate (EDANS)
360/720
2 mM bath
Fluo 4
Intracellular calcium
488/850
10 mM perfusate/bath
Fura Red
Intracellular calcium
( ratio = Ca2+ )
488/850
10 mM perfusate/bath
DAF-FM
Nitric oxide
495
10 mM perfusate/bath
BCECF
pH ( ratio = pH)
495/440
10 mM perfusate/bath
Nile Red
Neutral lipids
543/800
2 mM bath
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).
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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.
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.
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BASIC
PROTOCOL 5
Materials
C57 BL/6 mice or Munich-Wistar rats
Inactin (see recipe)
Ketamine
Krebs Ringer-HCO3 (see recipe)
Fluorescent probes (70-kDa rhodamine B conjugate, quinacrine, Lucifer Yellow)
Polyethylene tubing (0.86-mm i. d.)
Analog single-channel transducer signal conditioner (World Precision Instruments
model no. BP-1)
Two-photon laser scanning fluorescence microscope (e. g., Leica TCS SP2 AOBS
MP confocal microscope system) and HCX PL APO 63/1.4NA oil CS
objective lens (Leica)
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.
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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
Current Protocols in Cytometry
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.
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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
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)
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
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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
Adjust pH to 7.4 with 1 N NaOH or 1 N HCl
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
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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)
Adjust pH to 7.4 with 1 N NaOH or 1 N HCl
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 ).
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
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
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Molecular
Imaging
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Current Protocols in Cytometry
Supplement 44
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
12.12.24
Supplement 44
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
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
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Molecular
Imaging
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Supplement 44
Literature Cited
Denk, W., Strickler, J. H., and Webb, W. W.
1990. Two-photon laser scanning fluorescence
microscopy. Science 248:73-76.
Garaschuk, O., Milos, R.I., and Konnerth, A. 2006.
Targeted bulk-loading of fluorescent indicators
for two-photon brain imaging in vivo. Nat.
Protoc. 1:380-386.
Hwang, E. F., Williams, I., Kovacs, G., Peti-Peterdi,
J., Siroky, B., Rice, W. C., Bates, E., Schweibert,
E.M., Unlap, M. T., and Bell, P.D. 2003.
Impaired ability of the Na+/ Ca2+ exchanger
from the Dahl/Rapp salt-sensitive rat to regulate
cytosolic calcium. Am. J. Physiol. Renal Physiol. 284:F1023-F1031.
Kang, J. J., Toma, I., Sipos, A., McCulloch, F., and
Peti-Peterdi, J. 2006a. Quantitative imaging of
basic functions in renal (patho)physiology. Am.
J. Physiol. Renal Physiol. 291(2):F495-F502.
Kang, J. J., Toma, I., Sipos, A., McCulloch, F.,
and Peti-Peterdi, J. 2006b. Imaging the reninangiotensin system: An important target of antihypertensive therapy. Adv. Drug Deliv. Rev.
58:824-833.
From In Vitro to
In Vivo: Imaging
from the Single
Cell to the Whole
Organism
12.12.26
Supplement 44