The American Journal of Pathology, Vol. 179, No. 1, July 2011

Copyright © 2011 American Society for Investigative Pathology.

Published by Elsevier Inc. All rights reserved.

DOI: 10.1016/j.ajpath.2011.03.048

Biophysical Imaging and Computational Biology

Two-Photon Imaging within the Murine Thorax

without Respiratory and Cardiac Motion Artifact

Robert G. Presson Jr,*† Mary Beth Brown,*‡

Amanda J. Fisher,*† Ruben M. Sandoval,‡§

Kenneth W. Dunn,‡§ Kevin S. Lorenz,¶

Edward J. Delp,¶ Paul Salama,�

Bruce A. Molitoris,‡§** and Irina Petrache*‡**From the Center for Immunobiology,* and the Departments of

Anesthesia,† and Medicine,‡ Indiana University, Indianapolis; the

Indiana Center for Biological Microscopy,§ Indianapolis; the

School of Electrical and Computer Engineering,¶ Purdue

University, West Lafayette; the Department of Electrical and

Computer Engineering,� Indiana University–Purdue University,

Indianapolis; and the Richard L. Roudebush Veterans Affairs

Medical Center,�� Indianapolis, Indiana

Intravital microscopy has been recognized for its abil-ity to make physiological measurements at cellularand subcellular levels while maintaining the complexnatural microenvironment. Two-photon microscopy(TPM), using longer wavelengths than single-photonexcitation, has extended intravital imaging deeperinto tissues, with minimal phototoxicity. However,due to a relatively slow acquisition rate, TPM is espe-cially sensitive to motion artifact, which presents achallenge when imaging tissues subject to respiratoryand cardiac movement. Thoracoabdominal organsthat cannot be exteriorized or immobilized duringTPM have generally required the use of isolated,pump-perfused preparations. However, this ap-proach entails significant alteration of normal phys-iology, such as a lack of neural inputs, increased vas-cular resistance, and leukocyte activation. We adaptedtechniques of intravital microscopy that permittedTPM of organs maintained within the thoracoabdomi-nal cavity of living, breathing rats or mice. We ob-tained extended intravital TPM imaging of the intactlung, arguably the organ most susceptible to bothrespiratory and cardiac motion. Intravital TPM de-tected the development of lung microvascular endo-thelial activation manifested as increased leukocyteadhesion and plasma extravasation in response tooxidative stress inducers PMA or soluble cigarettesmoke extract. The pulmonary microvasculature and

alveoli in the intact animal were imaged with compa-

rable detail and fidelity to those in pump-perfusedanimals, opening the possibility for TPM of other tho-racoabdominal organs under physiological andpathophysiological conditions. (Am J Pathol 2011, 179:

75–82; DOI: 10.1016/j.ajpath.2011.03.048)

There is an increasing need for visualizing dynamic cel-lular and molecular events in the context of their naturalcomplex tissue environment. This task has long beenlimited by artifacts generated during tissue fixation or bychanges in cell behavior induced by ex vivo culture sys-tems. With the recent development of a wide variety offluorescent probes and transgenic expression of fluores-cent labels, intravital microscopy allows the dissection ofmolecular events with subcellular resolution in near realtime in the intact animal. Nevertheless, serious chal-lenges remain. For example, the use of routine epifluo-rescence microscopy for intravital imaging is limited by asuperficial plane of visualization and photodamage to thelipid- and water-rich structures of many biological tis-sues. Although confocal microscopy is capable of imag-ing structures at a deeper tissue level, the techniquesused to eliminate light arising outside the focal point (eg,pinhole aperture) inevitably degrade the signal whereasthe problem of phototoxicity remains. The development oftwo-photon excitation microscopy (TPM) has addressedsome of these limitations. In contrast to epifluorescenceand confocal microscopy, excitation of fluorescentprobes in TPM results from the simultaneous collision oftwo long-wavelength photons with the fluorophore. Be-cause excitation only occurs at the focal point, there is noout-of-plane fluorescence to reject, and a stronger signal

Supported by the National Institutes of Health (NIH)–National Heart, Lung,and Blood Institute grants R01 HL 077328 (I.P.); R21 DA029249-01 (I.P.and R.G.P.); T32 (M.B.B.); and NIH O’Brien P-30 grant 5P30DK079312.

Accepted for publication March 29, 2011.

R.G.P. and M.B.B. contributed equally to this work.

None of the authors disclosed any relevant financial relationships.

Supplemental material for this manuscript can be found at http://ajp.amjpathol.org or at doi: 10.1016/j.jmoldx.2010.11.003.

Address reprint requests to: Robert G. Presson Jr, M.D., or Irina Petra-che, M.D., Walther Hall–R3, C400, 980 W. Walnut Street, Indianapolis, IN

46202. E-mail: rpresson@iupui.edu or ipetrach@iupui.edu.

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76 Presson Jr et alAJP July 2011, Vol. 179, No. 1

can be acquired. Moreover, the lower-energy excitationphotons in TPM are less likely to produce phototoxicityand are able to penetrate farther into tissue, thus permit-ting examination into organs of living animals. Unfortu-nately, the relatively slow rate of image acquisition makesTPM especially sensitive to motion artifact, which pres-ents a major obstacle to imaging tissues subject to re-spiratory and cardiac movement. Motion artifact can becircumvented for organs, such as the kidney1 and liver,2

that can be surgically explanted from the body for imag-ing without alteration in perfusion or organ function. Tho-racoabdominal organs that cannot be exteriorized or im-mobilized during TPM, such as the lung, have generallyrequired the use of isolated, pump-perfused prepara-tions.3–5 However, measurements obtained from isolatedpreparations may vary from the intact animal (that main-tains tissue perfusion by means of a beating heart) for anumber of reasons, including an absence of neural in-puts, increased vascular resistance, and activation ofleukocytes by foreign surfaces of the perfusion circuit.4

Motion can be largely eliminated in intact preparations forintravital imaging by suspending ventilation during acqui-sition,6 but the duration of apnea can generally not belong enough for image capture in more than one plane,given TPM’s relatively slow framing rate. Suspendingventilation is also not an ideal solution to motion artifact inlung imaging because of potentially confounding effectsintroduced by altered blood gases and pulmonary andsystemic pressures. Here, we describe novel adaptationsof previously pioneered intravital microscopy tech-niques4,7 that permit high-fidelity imaging of lung main-tained within the thoracoabdominal cavity of a living,breathing animal for a prolonged period of time. Our workcomplements two very recent reports that also describethe innovative use of intravital lung TPM in intact rodentmodels.8,9 In addition, we present the first detailed meth-odology for management of motion artifact to obtain con-sistent, high-quality three-dimensional and time-lapseTPM imaging of murine lung under physiological condi-tions and document its superiority to the pump-perfusedpreparation in maintaining physiological cardiopulmo-nary hemodynamic and biochemical parameters.

Materials and Methods

Stabilization at the Lung–Microscope Interface

To perform TPM of the lung in the intact rat, we optimizedthe lung–microscope interface with an imaging windowuniquely designed to minimize cardiac and respiratorymotion while permitting the use of high numerical aper-ture TPM objectives (see Supplemental Figure S1 athttp://ajp.amjpathol.org). The window was located in thecenter of a purpose-made acrylic tray (see SupplementalFigure S1A at http://ajp.amjpathol.org), constructed forintravital microscopy of organs within the thoracoabdomi-nal cavity, that was mounted on the microscope stage ofthe TPM system. The window design consisted of a cor-rosion-resistant aluminum frame (see Supplemental Fig-

ure S1, B and C, at http://ajp.amjpathol.org) holding a

circular coverslip 18 mm in diameter. The coverslip wassurrounded by a vacuum ring that held the lung in con-tact with the coverslip and reduced respiratory move-ment, allowing observation of a 0.5 cm2 area of the over-lying lung (see Supplemental Figure S1D at http://ajp.amjpathol.org). A similar design was adapted toimage the mouse lung.

Intact Animal Preparation

All animal studies were conducted in compliance with theInstitutional Animal Care and Use Committee guidelinesof Indiana University. Adult male Sprague Dawley rats(Harlan, Indianapolis, IN) weighing 250 to 350 g wereanesthetized by inhaled isoflurane (5% in oxygen) andorotracheally intubated with a 6-F catheter. The animalswere placed on a servo-controlled heating pad, and theleft carotid artery and right external jugular vein werecannulated via cutdowns. Surgical instruments were au-toclaved before each use to minimize the introduction offoreign pathogens. The lungs were ventilated using atidal volume of 6 mL/kg, a rate of 60 breaths/minute, aninspired O2 content of 100%, and isoflurane to maintaingeneral anesthesia. Airway pressure and esophagealtemperature were monitored and maintained continu-ously at 14 to 19 cm H2O (peak), and 36 to 37 °C,respectively. End-expiratory pressure was maintained at�5 cm H2O. Blood gases were sampled periodically,respiratory rate was adjusted to maintain a pACO2 of 35to 40 torr, and inspired oxygen concentration was ad-justed to maintain a pAO2 � 90 torr. The systemic arterialpressure was monitored continuously, and lactated Ring-er’s solution was infused via the venous catheter at amaintenance rate of 3 mL/kg/hour with boluses given asneeded to replace blood loss from sampling. The animalswere placed in the right lateral decubitus position, and a6-cm-long skin incision was made 2 cm caudal to theposterior edge of the left scapula. The latissimus dorsiwas retracted posteriorly, and a thoracotomy was madein the left fifth intercostal space. Stay sutures were placedaround the anterior and posterior ends of the fifth andsixth ribs approximately 2 cm apart (see SupplementalFigure S2 at http://ajp.amjpathol.org). The thoracotomywas held open by tension on the stay sutures, and theanimals were placed in the left lateral decubitus positionwith the left lung centered over the window. End-expira-tory pressure was briefly increased to 10 to 20 cm H2O,which brought the lung into contact with the coverslip. Avacuum (�40 mm Hg) was applied to the window thatheld the lung in position after the end-expiratory pressurewas returned to 5 cm H2O, a level typically used formechanical ventilation, which does not adversely affectmicrocirculation.10 A similar procedure was performed inmice with the following modifications: Mice were orotra-cheally intubated with a 20-gauge catheter and ventilatedat a rate of 130 breaths/minute. The right internal jugularvein was cannulated via cutdown with a 26-gauge cath-eter for administration of fluid and fluorescent probes. Athoracotomy was performed in the fifth left intercostal

space, and the sixth rib was excised. The window, mea-

Intravital Microscopy of the Intact Lung 77AJP July 2011, Vol. 179, No. 1

suring 1 cm in diameter, was interfaced with the lung viathis thoracotomy, as described for the rat.

Pump-Perfused Lung Preparation

Because respiration can be suspended for long periodswithout altering blood gases and because there is nocardiac motion, we also performed TPM imaging of thelung in the isolated pump-perfused preparation for com-parison to the images acquired from the intact rat. Sur-gical procedures for the pump-perfused lung preparationwere similar to the procedures for the intact preparationwith a few exceptions. For the pump-perfused prepara-tion, following orotracheal cannulation, carotid cannula-tion, and exsanguination, the blood was added to a per-fusion circuit already primed with buffer (20 mmol/LHEPES, 125 mmol/L NaCl, 3.5 mmol/L KCl, 1 mmol/LCaCl2, 1 mmol/L MgCl2, 5 mmol/L glucose, and 1% fetalbovine serum, final hematocrit �10%, circulating volume�30 mL). The left chest wall was excised, and the apex ofthe heart was transected. The arterial perfusion cannulawas passed through the right ventricle across the pul-monic valve into the main pulmonary artery and wassecured with a ligature passed around the main pulmo-nary artery and aortic root. The venous perfusion cannulawas passed through the left ventricle across the mitralvalve into the left atrium and was secured with a ligaturearound the atrioventricular groove. Ventilation was re-sumed with 5% CO2 in air, and perfusion was initiated ata flow rate of 5 mL/min. Blood was pumped (Gilson Mi-nipuls 3; Gilson, Middleton, WI) through a heat exchangerinto the pulmonary artery and drained passively from theleft atrium into a reservoir. Pump flow rate was slowlyincreased to maintain a mean arterial pressure �15 mmHg, and the height of the reservoir was adjusted to obtaina venous pressure of �2 mm Hg. Animal positioning andthe regulation of blood gases, esophageal temperature,and pressures for intravital microscopy were performedas described for the intact preparation.

Fluorescent Conjugates

Rat serum albumin (RSA; Sigma-Aldrich, St. Louis, MO)or a 150-kDa amino dextran (TdB Consultancy, Uppsala,Sweden) was conjugated to fluorescein isothiocyanate(FITC) or Texas Red (Invitrogen, Carlsbad, CA) as out-lined in the manufacturer’s technical instructions foramine reactive probes (Molecular Probes/Invitrogen). Af-ter conjugation of FITC or Texas Red to the RSA, anyunbound dye molecules were removed by dialyzingagainst 0.9% NaCl in double-distilled H2O using a 300-kDa cutoff MWCO filter (Cellulose Ester Membrane fromSpectrum Laboratories, Rancho Dominguez, CA). EitherFITC-RSA (12 to 14 mg/kg) or similarly labeled FITC- orTexas Red–Dextran (150 kDa amino dextran; 20 to 22mg/kg) were administered intravenously (i.v.) to label thecirculating plasma. Nuclei were stained with Hoechst33258 (10 to 12 mg/kg; i.v.; Invitrogen), and leukocyteswere labeled with Rho-G6 (0.2 mmol/L in saline; 0.3 mL/

kg; i.v.; Sigma-Aldrich).

Additional Motion Reduction Techniques

Although the vacuum ring markedly reduced respiratoryand cardiac-induced lung motion, in some cases, addi-tional procedures were needed to obtain high-fidelity im-ages without interruption of ventilation. Therefore, we de-veloped two approaches to deal with these challenges:gated imaging and frame registration. In gated imaging,Z-stack acquisition is synchronized (gated) to the respi-ratory cycle for “between breath” image capture. Gatedimaging was accomplished by communication betweena laptop computer running LabVIEW software (NationalInstruments Corporation, Austin, TX) that controlled theventilator and a second computer running the imagingsoftware (Fluoview 2.1c; Olympus, Center Valley, PA) viaa DB15 interface. Briefly, the ventilator was modified tosend a 5-V signal to the laptop computer at the start ofexpiration. At that point, the laptop paused the ventilatorvia a relay and then signaled the imaging computer tostart a scan. After completion of the scan (�2 seconds),a signal was sent to the laptop that closed the relay,resuming ventilation. This cycle was repeated until thedesired number of scans was obtained.

Due to a lower frequency of image acquisition, gatinginherently decreases temporal resolution. Therefore, toobtain high-fidelity images at high speed when cardiacand respiratory motion was not sufficiently reduced bythe vacuum window, a novel frame registration methodwas applied in image processing. Motion artifacts werereduced postcollection using a novel nonrigid image reg-istration method that uses B-splines to correct for motionartifacts occurring in images collected in time series or inthree-dimensional volumes. The method involves creat-ing a uniform grid of control points in the images of thetime series, each of which is manipulated by optimizing acost function based on image similarity and grid smooth-ness. The technique is described more fully in Lorenzet al.11

Two-Photon Microscopy System

Unless otherwise specified, TPM was conducted on aBio-Rad MRC 1024 confocal/two-photon system (Bio-Rad, Hercules, CA) mounted on a vibration-isolation table(see Supplemental Figure S3 at http://ajp.amjpathol.org).The illumination source was a tunable Tsunami Ti:Sap-phire laser (Spectra-Physics, Mountain View, CA). Themicroscope was an inverted Nikon Eclipse TE200 (Nikon,Melville, NY). The band-pass filters used for the red andgreen channels were 605/690 nm and 525/550 nm, re-spectively. The excitation wavelength was set at 800 nmfor all experiments. Either a �60 or a �20 Nikon Plan Apowater immersion objective (NA 1.2 and 0.8) was used forimaging and was warmed with an objective heater (War-ner Instruments, Hamden, CT). All image series werecollected at a constant pixel dwell time, which yielded a1.1-second frame time for the frame size of 512 � 512

pixels (169 � 169 �m).

78 Presson Jr et alAJP July 2011, Vol. 179, No. 1

Cigarette Smoke Soluble Extract

Filtered research-grade cigarettes (1R3F) from the Ken-tucky Tobacco Research and Development Center (Uni-versity of Kentucky, Lexington, KY) were used for prepar-ing aqueous cigarette smoke extract. Cigarette smoke(100%) was prepared by bubbling smoke from four cig-arettes into 5 mL of PBS at a rate of one cigarette/minuteto 0.5 cm above the filter,12 followed by pH adjustment to7.4 and 0.2-�m filtration.

Measures of Microvascular Flow and LeukocyteAdhesion

Microvascular flow was determined for horizontally ori-ented vessels as previously described,13 using vesseldiameter and blood velocity as calculated by slope of theblack streak that appears where red blood cells are im-aged in motion. Stationary Rho-G6–labeled intraluminalleukocytes adherent to the microcirculation within a sin-gle field were counted using frames of time-series mov-ies, as previously described.14

Statistical Analyses

Statistical analyses were performed using SigmaStat 3.5.Comparisons among groups were made using analysisof variance followed by multiple comparisons versus acontrol group (Holm-Sidak method). For experiments inwhich two conditions were being compared, a two-tailedStudent’s t-test was used. All experiments were per-formed at least three times. All data are expressed asmean � SEM, and statistically significant differenceswere considered if P � 0.05.

Results

TPM Imaging of the Lung

Mild suction incorporated into a custom-made imagingwindow effectively eliminated cardiac and respiratorymotion at the lung–microscope interface in most intactpreparations for high-quality TPM. Single-frame imagesfrom time-series acquisition movies in the intact rat underphysiological conditions (during ventilation, heart per-fused) are shown in Figure 1, illustrating the three mainvessel types captured by TPM: pulmonary arterioles,venules, and capillaries. For both the intact rat and theintact mouse, intravenous nuclear staining revealed pri-marily endothelial cells lining the vessels in images ob-tained by Z-stack (see Supplemental Video S1 in rat andVideo S2 in mouse at http://ajp.amjpathol.org) or time-series acquisition (see Supplemental Video S3 in rat andVideo S4 in mouse at http://ajp.amjpathol.org). When theintravenous administration was combined with intratra-cheal delivery, the nuclear dye penetrated deep in theparenchyma, providing staining of additional nuclei, seenin the three-dimensional reconstruction in SupplementalFigure S4 (available at http://ajp.amjpathol.org). Based on

location and size, these additional nuclei were likely con-

tained within alveolar epithelial cells and alveolar macro-phages.

Images from the intact animal were equal in detail andfidelity to those obtained for comparison in the pump-perfused preparation, in which respiratory and cardiacmotion is not a factor (Figure 2). In either the intact animalor the pump-perfused preparation, the average numberof alveolar spaces captured in a single field of view was38 � 4 (with the �60 objective) or 62 � 8 (with the �20objective), at a maximum depth ranging from 40 to 60 �mfrom the pleural surface.

Gating Imaging and Frame Registration

In some cases, where lung–microscope stabilization pro-vided by the vacuum ring could not completely eliminaterespiratory and cardiac-induced lung motion, gated im-aging or frame registration was additionally used. Gatedimaging was used for Z-stack acquisition and entailedcomputerized synchronization of the respiratory cycle toimage capture, effectively eliminating motion and maxi-mizing clarity of anatomical detail in single-plane andreconstructed three-dimensional images (Figure 3). Al-though the gating algorithm decreased the overall respi-ratory rate, it did not significantly change the arterialblood gases (see Supplemental Table S1 at http://ajp.amjpathol.org). For time-series collections, frame registra-tion11 was used in postcapture image processing to cor-rect for motion artifact when necessary and effectivelyremoved the rhythmic lung motion that was captured in

Figure 1. Representative frames of time-series TPM images in the intact ratshowing FITC-labeled (green) pulmonary microvasculature in low magnifi-cation �20 (A) and high magnification �60 (B–D). Note detailed microvas-culature morphology of medium- and small-sized arterioles (white arrow-heads, A), venules (white bidirectional arrows, C and D), and capillaries(yellow arrows) surrounding normal alveolar airspaces. Nuclei are stainedwith intravenous Hoechst (blue), and circulating cells appear as black streakswithin the vessels. Scale bars: 75 �m (A) and 25 �m (B–D).

some movies acquired during ventilation, without com-

Intravital Microscopy of the Intact Lung 79AJP July 2011, Vol. 179, No. 1

promising temporal resolution (see Supplemental VideoS3 at http://ajp.amjpathol.org).

Quality of the Physiology

Intact rats were hemodynamically stable during experi-ments, which averaged 6 to 7 hours, from the time of

Figure 2. Three-dimensional reconstruction of FITC-labeled microvascula-ture (green) surrounding normal alveolar airspaces (dark regions) imaged inintact preparations (A and C) are comparable to pump-perfused preparations(B and D). Nuclei are stained blue with intravenous Hoechst. Scale bars � 25�m. Note that the capillary circulation (A–D) and larger venules (C and D) arevisualized by TPM with similar detail in the two preparations.

Figure 3. Gated imaging eliminates motion for maximum clarity in three-dimensional TPM reconstructions. A: Comparison between ungated (left)and gated (right) image acquisition of an identical field of view showingFITC-labeled (green) alveolar microvasculature in the intact rat. Reconstruc-tions in the x-z orientation correspond to the indicated slice regions (B–E)

from the 3 dimensional images. Nuclei are stained with intravenous Hoechst(blue). Scale bars � 25 �m.

initiation of surgical preparation to placement on the mi-croscope (2 to 3 hours), to the completion of intravitalimaging (3 to 4 hours). Peak arterial blood pressure wasmaintained above 90 mm Hg; and mean partial pressuresof oxygen (pAO2) and carbon dioxide (pACO2) weremaintained in the range of 90 to 100 mm Hg (fraction ofinspired O2, 0.21 to 0.30) and 35 to 45 mm Hg, respec-tively (Table 1). Vessel diameter and microvascular flowwere stable over the period of observation (see Supple-mental Table S2 at http://ajp.amjpathol.org). Further, mi-crovascular flow was better maintained in pulmonary ar-terioles, venules, and capillaries of the intact animalcompared to those imaged in the in the pump-perfusedlung (Table 2). The significantly lower microvascular flowin the pump-perfused lung is due to the elevated pulmo-nary vascular resistance characteristic of this prepara-tion.15 This necessitated a low pump flow rate (to main-tain perfusion pressures in the normal range), even with ahemoglobin concentration of 4 g/dL. In aggregate, thesedata show that physiological parameters were main-tained over an extended period of experimental observa-tion without tissue damage in the intact rat preparation.

Applications to Studies of Lung Pathology

The novel adaptation of intravital imaging methods de-scribed above can be used to investigate in vivo organphysiology, signaling, pharmacodynamics, and disease

Table 1. Vital Signs and Arterial Blood Parameters during TPMin the Intact Animal

Systolic arterial pressure, mm Hg 100 � 8Diastolic arterial pressure, mm Hg 62 � 12Heart rate, beats/minute 350 � 44Arterial pCO2, mm Hg 42 � 8Arterial pO2, mm Hg 110 � 18Arterial pH 7.42 � 0.04Hematocrit, %PCV 34 � 6Hemoglobin, g/dL 12 � 2

These values are within physiological range and are presented asmean � SD; n � 11.

PCV, packed cell volume; TPM, two-photon microscopy.

Table 2. Microvasculature Hemodynamic Parameters in the Intact(Live) Animal Compared to Those of the Pump-PerfusedPreparation during Intravital TPM

Flow (mm3/second)

Intact prep(n � 11)

Pump-perfusedpreparation

(n � 6)

Medium arteriole(19–31 �m)

580 � 300 430 � 170

Small arteriole (10–18 �m) 140 � 90* 40 � 9Medium venule (19–28 �m) 640 � 380** 200 � 90Small venule (10–18 �m) 150 � 80*** 85 � 48Capillary (6–9 �m) 31 � 16*** 19 � 8

Values are the mean � SD of hemodynamic parameters recordedduring 3 hours of experiments.

*P � 0.05, **P � 0.01, and ***P � 0.001; all versus pump-perfused

preparation.

TPM, two-photon microscopy.

v; 5 to 2� SD; *

80 Presson Jr et alAJP July 2011, Vol. 179, No. 1

pathogenesis. For example, we captured the dynamicresponses of the lung microvasculature to perturbationsinduced by the intravenous administration of the oxida-tive stress and tumor promoter 4b-phorbol 12-myristate13-acetate (PMA; Santa Cruz Biotechnology, Santa Cruz,CA; 1 mmol/L in dimethylformamine). PMA is known toactivate lung macrophages and result in irreversible pa-renchymal damage via destruction of endothelial andalveolar epithelial cells of the distal airways. Breach inalveolar and microvasculature barrier function from PMAadministration results in alveolar hemorrhage and flood-ing.16 In these experiments, intravenous rhodamine 6G(Rho-6G; 0.5 mg/kg) was used to label leukocytes for fluo-rescence imaging (Figure 4; see also Supplemental VideoS5 at http://ajp.amjpathol.org). In response to PMA admin-istration, leukocytes accumulated in the lung paren-chyma within 5 minutes and demonstrated reduced mo-bility through the microcirculation, suggesting increasedadhesion to the endothelium (Figure 4C; see also Sup-plemental Video S6 at http://ajp.amjpathol.org). In addi-tion, we recorded progressive breaching of the lungmicrovascular barrier signaled by extravasation of FITC-RSA into the alveolar airspaces (Figure 4, D–F; see alsoSupplemental Video S6 at http://ajp.amjpathol.org), con-sistent with the development of pulmonary edema. Theearly development of endothelial activation, manifestedas increased leukocyte adhesion to the microvasculaturecan be quantified using a technique we previously ap-plied for the kidney microcirculation.14 We noted a sig-nificant increase in leukocyte adhesion starting at 5 min-utes following PMA administration (Figure 4). A similareffect was induced by administration of soluble compo-nents of cigarette smoke (Figure 4), known to induce

Figure 4. Capturing real-time development of pulmonary edema using TPMFITC-labeled vessels (green) surrounding alveoli without (A) and with Rho-6Gadministration of PMA (1 mmol/L, 0.10 mg/kg). Nuclei are stained with intravcapillaries at 5 minutes (C; arrow) and plasma extravasation (asterisks)administration. D and E depict an identical field of view. Scale bars � 25 �min vivo in response to PMA (1 mmol/L in dimethylformamine, 100 �L/kg, i.minutes) administration compared to untreated animals (n � 2 rats). Mean

oxidative stress by highly diffusible free radicals that

cause microvascular activation in vitro (Wagner17 andunpublished data).

Discussion

We have described an innovative method of intravitalmicroscopy that permits high-resolution subsurface im-aging of organs within the thoracoabdominal cavity of aliving animal without interference from cardiac or respi-ratory motion. First, we used a vacuum ring imagingwindow, custom-designed for the TPM optics that phys-ically restrained tissue at the organ–microscope inter-face. Second, we applied gated imaging or frame regis-tration as adjunctive approaches to optimize imagequality for situations in which the vacuum ring alone didnot sufficiently immobilize the observed tissue. Themethod of gating imaging to the respiratory cycle en-abled capture of a series of planes in the z-direction“between breaths” for use in three-dimensional imagereconstructions. Alternatively, frame registration applieda postacquisition algorithm to correct for rhythmic respi-ratory motion when maximal temporal resolution was re-quired. These highly detailed, high-quality and stableTPM images of the lung, an organ inexorably coupled toboth cardiac and respiratory movement, obtained inphysiological conditions, are evidence of the power ofthese techniques for imaging thoracoabdominal organsin situ. Early attempts to deal with the problem of motionduring intravital imaging of the lung in the intact animalincluded simply suspending respiration,6 with observa-tions made during apnea. Major progress toward physi-ological imaging of the lung was made by Wagner,7 who

lung microvasculature in the intact rat. Three-dimensional reconstruction ofd leukocytes (red, B–F) imaged before (A and B) and after (C–F) intravenousadministered Hoechst (blue). Note increasing leukocyte sequestration in the

rspaces at 9 minutes (D), 12 minutes (E), and 30 minutes (D) post-PMAsentative image of n � 3 animals. G: Quantification of leukocyte adherence0 minutes) or soluble cigarette smoke extract (100%, 2 mL/kg i.v; 20 to 30

P � 0.001 versus control; n � 5 to 19 time-lapse movies.

of the–labele

enouslyinto ai. Repre

implanted a device in the chest wall containing a central

Intravital Microscopy of the Intact Lung 81AJP July 2011, Vol. 179, No. 1

window and a surrounding vacuum manifold that ar-rested cardiorespiratory motion in the observation fieldduring serial measurements. Although this approach per-mitted bright-field imaging of the subpleural microcircu-lation in a living, breathing animal, observations werelimited to less than a few microns from the surface. Later,fluorescence microscopy was applied using a similarwindow,18 and conventional confocal microscopy wasused to visualize alveoli deeper than those accessible towide-field microscopy.19,20 However, these approacheswere performed only in isolated (pump-perfused) lungpreparations. Recently, the use of a probe inserted trans-diaphragmatically or transthoracically allowed the use ofthe confocal microscopy in the intact animal.21 However,the confocal aperture’s rejection of scattered light leadsto a relatively weak signal that may render a poor imagequality, and no quantitative or dynamic time-lapse anal-yses during the ventilatory motion were reported. WithTPM, there is no out-of-plane fluorescence to reject, andtherefore, no confocal aperture to obstruct photon collec-tion. The resulting signal is stronger, as shown in a pump-perfused preparation,4 and in our hands, was capable ofimaging structures as deep as 80 �m into the lung withgood resolution. Within these parameters, we demon-strated that both alveolar capillary structures and largervenules and arterioles can be adequately visualized inreal time while maintaining physiological parameters forover 3 hours of study. The quality of the preparation andits performance surpassed that of the pump-perfusedpreparation, while simplifying and shortening the surgicalpreparation time and trauma to the animal. Althoughmeasures were taken to minimize contamination by for-eign pathogens during each animal preparation, a poten-tial risk of any surgery as invasive as required for intravitalimaging within the thorax is that nonsterile conditions mayaffect leukocyte behavior at the imaging site or otherendpoints of analysis. Although our control imaging stud-ies of the lung microcirculation over the entire duration ofthe experiment (up to 3 hours) did not reveal markedincreases in polymorphonuclear leukocyte accumulationor adherence to the microendothelium, our method canbe further improved by ensuring total sterility of the sur-gical field and of the animal–microscope interface. Twoindependent reports of intravital TPM to investigate lunginflammation in rodents8,9 have been published in thepast months, highlighting the importance and emergenceof this method for the study of complex integrated re-sponses in the lung. To complement these studies, weprovide a detailed and reproducible protocol of intravitalTPM of the lung that generates detailed, stable, high-fidelity three-dimensional and time-lapse imaging of themurine lung microcirculation and alveolar spaces; weapply for the first time noninvasive approaches to stabi-lize the intravital tissue imaging (avoiding the use of su-perglue or breath-hold to immobilize the lung to the im-aging window); and we document the preservation offunctional parameters such as cardiopulmonary hemody-namics and biochemical homeostasis in the animal forthe duration of the experiment (up to 3 hours).

The adapted intravital methods described herein have

a vast breadth of application within medical research,

particularly for real-time visualization of processes withinthoracoabdominal organs without significant disruption ofthe normal physiological milieu. We demonstrated a rapidand sensitive capture of intra-alveolar edema develop-ment and leukocyte sequestration in pulmonary capillar-ies following infusion of PMA, a typical oxidative stressinducer. Leukocyte adhesion could be quantified andallowed sensitive detection of endothelial cell activation,consistent with early inflammatory changes in responseto both PMA and exposure to soluble components ofcigarette smoke. Such changes could not be accuratelydetected in pump-perfused preparations, which areknown to activate leukocytes by exposure to foreign sur-faces. We also used our imaging technique to calculateblood flow in various pulmonary vascular structures.These are just a few examples of applications of our methodto study tissue physiology and pathology, which could beexpanded to include studies of vasoreactivity22; endothelialor epithelial permeability23,24; leukocyte adherence and roll-ing8,9,14; cell injury and apoptosis25; endo-, trans-, and exo-cytosis26,27; ionic gradients28; metabolic status29; intracel-lular trafficking and subcellular localization26; and drugdeposition and biodistribution.30,31

Continued development of improved probes amenableto visualization of rapid events in living animals will ad-vance the application of TPM to investigations of complexprocesses such as intra- and intercellular signal trans-duction. These mechanistic studies will be further facili-tated by the use of genetically modified mice that usetransgenically expressed functional probes. In conclu-sion, we describe a novel method of TPM of the lungparenchyma in a live rodent, which can be applied to anyorgan prone to motion artifact to study physiological andpathological processes in their natural, unperturbed, andinterconnected environment.

Acknowledgments

We thank George Rhodes, M.D., for technical assistancewith the microscope, and Jason Byars, who providedassistance with programming for gated imaging.

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