Many diseases of the eye, especially those that causeinner retinal neovascularization (diabetic retinopa-thy, retinopathy of prematurity, sickle cell disease,etc.) and those that involve retinal degeneration (ret-initis pigmentosa, age-related macular degeneration)have regional hypoxia as either a primary causativeor an early contributory factor. Regions of hypoxiaare likely to appear before irreversible tissue injuryoccurs. This is particularly true for diabetic retinop-athy, the leading cause of blindness for individualsfrom 20 to 74 years of age. Multiple structures of theeye are pathologically affected in diabetics by neovas-cular changes with resultant plasma leakage andtissue disruption.1 Currently available data are con-sistent with the hypothesis that hypoxia of the retinais responsible for pathologic growth of new blood ves-sels in the inner retina, the major cause of blindnessassociated with diabetes of long duration. Support forthis idea includes oxygen electrode measurements byLinsenmeier and co-workers,2,3 showing that in dia-betic cats the oxygen pressures in the inner half of theretina are approximately one half of those in normal
cats, and by Berkowitz et al.4, showing that there wasdecreased oxygen response in the retinas of galac-tosemic rats. Oxygen electrodes are useful in identi-fying global oxygen deficiency, but they are invasiveand not highly effective for assessing hypoxia inducedby failure of individual capillaries. Retinal pathol-ogy in diabetes, however, is believed to result fromprogression of intraretinal microangiopathy (nonpro-liferative diabetic retinopathy) to extraretinal neo-vascularization.5 Similar pathogenic mechanisms arethought to occur in other diseases characterized byinner retinal neovascularization. Small, focal areas ofhypoxia resulting from clusters of defective capillar-ies could be responsible for production of high levelsof vascular endothelial growth factor (VEGF). Vitre-ous samples in human beings with retinopathy havebeen shown to contain high levels of VEGF.6 Highlevels of VEGF have also been measured in animalmodels of ocular neovascular disease.7–10 VEGF,in turn, has been implicated as a mediator of newblood vessel formation. Recently, treatments for age-related macular degeneration have been proposedbased on the premise that increased VEGF in the eyeis responsible for the observed vascular proliferation.Clinical trials are under way, for example, to test theefficacy of monthly injections of anti-VEGF aptamer(Eyetech Pharmaceuticals, New York), which bindsand inactivates VEGF, in suppressing developmentof vascular abnormalities.
Oxygen-dependent quenching of phosphores-cence11–18 appears to be well suited for measuringoxygen in the retinal vessels. It is minimally invasive,requiring only intravenous injection of a phosphor,
D. F. Wilson ([email protected]) S. A. Vinogradov,P. Grosul, and M. N. Vaccarezza are with the Departments ofBiochemistry and Biophysics and A. Kuroki and J. Bennett arewith the Department of Ophthalmology, School of Medicine, Uni-versity of Pennsylvania, Philadelphia, Pennsylvania 19104.
and phosphors have become available that are watersoluble and nontoxic. The lens of the eye readilytransmits light of both excitation and emission wave-lengths. By use of intensified CCD cameras of suffi-cient sensitivity, oxygenation of the vessels of theretina can be imaged by phosphorescence with goodspatial resolution. However, until recently oxygenmaps were obtained only for the relatively large cat orpig eyes.13,14,16,19 Recently Shonat and Kight17,18 suc-ceeded in obtaining images of oxygen distribution inthe mouse eye by using phosphorescence-lifetime im-aging. In this paper we demonstrate that phosphores-cence imaging can effectively define small vascularlesions, determining both the size and the severity ofthe local region of hypoxia as well as revealing indi-vidual retinal capillaries with abnormally low oxygenlevels.
2. Methods and Materials
A. Phosphorescence-Lifetime Imaging
The phosphorescence-lifetime imaging system previ-ously used to image oxygen in the retina of the cateye13,14 was modified to permit imaging of phospho-rescence lifetimes in the much smaller mouse eye.Following the lead of Shonat and Kight,17,18 weswitched to a frequency-domain approach. For clar-ity, the principles of lifetime imaging in the frequencydomain are briefly summarized below.
In a conventional frequency-domain lifetime mea-surement experiment (see, for example, Ref. 20), theexcitation source, e.g., a LED, is modulated by a si-nusoidal wave of frequency f:
Ex(t) � A sin(2�ft) � B, (1)
where Ex�t� designates the intensity of the excitationlight and A and B are the amplitude and the dc offsetof the excitation sine wave, respectively. The phos-phorescent response to the sinusoidal excitation[Em�t� is also a sinusoid of the same frequency (f) butdelayed in time �t] results in phase shift �:
Em(t) � a sin(2�ft � �) � b, � � 2�f � �t, (2)
where a and b are the amplitude and the dc offset ofthe phosphorescent signal. The value of the phaseshift is related to phosphorescence lifetime � by asimple relationship:
tan(�) � 2�f�. (3)
Determination of lifetime �, therefore, requires col-lecting signal Em�t� in digital form, finding phaseshift �, and converting it to � by using Eq. (3).
In imaging, it is conventional to use a simplifiedversion of excitation, i.e., to turn the excitation sourceon and off, producing a sequence of square pulses(Fig. 1). In addition, instead of collecting the entireresponse Em�t�, the detector, i.e., the CCD camera, isalso modulated by on–off switching at the same fre-
quency as the excitation but with a delay T. As aresult, the integrals of phosphorescent decays thatcorrespond to each pixel are acquired on the CCDarray. The excitation square wave �P�t�� can be pre-sented as a sum of fundamental frequency f and all itsharmonics:
P(t) � A �n�1
sin(2�nft) � B. (4)
The resultant phosphorescence is also the sum ofsinusoids, each with its own amplitude an, phase shift�n, and time delay �tn:
P(t) � �n�1
an sin(2�nft � �n) � b, � � 2�nf � �tn.
(5)
The phosphorescence image �I�T�� formed on theCCD array, modulated with delay T, is given by theintegral
I(T) � C�T
1�f
��n�1
an sin(2�nft � �n)dt, (6)
Fig. 1. Images of the phosphorescence intensity from the retina ofthe mouse eye. The mouse was treated as described in Section 2,and the phosphorescence intensity images were collected at1030 Hz. The number on each image indicates the delay (in de-grees) relative to the excitation for which the image was collected.The retinal area imaged is approximately 0.92 mm high by 1.2 mmwide.
where the upper limit of integration �1�f� representsthe period �360°� of the excitation square wave offundamental frequency f and constant C is related tothe acquisition time. It can be shown that the depen-dence of image intensity I on delay time T can beclosely approximated by the cosine form
I(T) c � cos(2�fT � �) � d, (7)
where � is the phase delay that corresponds to fun-damental frequency f used for modulation and c andd are constants. Thus, one can determine phospho-rescence lifetime � in each pixel by collecting a se-quence of images I�T� at different delays T, fittingthese images by using Eq. (7) and determining phaseshift � for each pixel, and converting phase shift � tolifetime � in each pixel, using Eq. (3). Obviously, dif-ferent modulation frequencies will result in differentphase delays for the same lifetime �. As a result ofsignal-to-noise ratio considerations, optimal phasedelays for determination of lifetimes are in the range20°–40°. If oxygen pressures and lifetimes in the im-age vary significantly, it is useful to produce the life-time maps at several modulation frequencies (seebelow), making the phase delays that correspond tovarious lifetimes fit into the optimal range ��30°�.
In our experimental setup the retina was observedunder a microscope with long-working-distance�18 mm� microscope lenses and on-axis illuminationthat we achieved by placing a dichroic mirror in theoptical path. The dichroic mirror reflected the excita-tion light from a modulated source (LED), positionedat a right angle relative to the optical axis of themicroscope, onto the retina while it transmittedthe longer-wavelength phosphorescence back alongthe axis to the CCD camera. Optical filters were usedto complete the separation of the excitation from thephosphorescence, which could be done with excellentefficiency because of very large Stokes shifts (morethat 150 nm) of the phosphorescence relative to theabsorption.
A Xybion (now ITT Night Vision, Roanoke, Va.)ISG 750 camera with enhanced red sensitivity wasused for detection of the phosphorescence. The inten-sifier of the camera can be turned on or off (gated) inapproximately 100 ns, and the excitation LEDs haveresponse times of less than 1 �s. Both the cameraintensifier and the LEDs could be modulated at fre-quencies from 100 Hz to 40 kHz. Phosphorescenceintensity images were collected at the modulationfrequencies that generated 15°–40° phase delay ofthe phosphorescence relative to the excitation. Forthe phosphors used and the oxygen concentrationsthat are typically present in vivo, this collectionrequired modulation frequencies in the range 400–3000 Hz. To calculate phosphorescence lifetimes, wecollected phosphorescence-intensity images at 6 to 15delays and analyzed them as described above.
Quenching of phosphorescence by oxygen followsthe Stern–Volmer equation
�0�� � 1 � kQ � �0 � pO2, (8)
where �0 and � are the phosphorescence lifetimes inthe absence of oxygen and at oxygen pressure pO2,respectively, and kQ is a second-order rate constantrelated to the frequency of collision of excited-statephosphor molecules with oxygen. Equation (8) is usedto convert the lifetime image into the oxygen image.
In spite of optical filtering, phosphorescent signalscollected from tissue are often mixed with reflectedexcitation light, endogenous fluorescence, or both. Thefluorescence signals have no delay with respect to theexcitation (in phase), and when they are added to ormixed with the phosphorescence they shorten the ap-parent lifetimes. One can remove in-phase signalsfrom the intensity images by collecting additional im-ages at 0° and 180° relative to the excitation usingsufficiently high frequencies. At high frequencies, al-ternating components of signals that correspond tolong lifetimes �� � 20 �s� are greatly suppressed; i.e.,the signals become practically constant in time (dc).However, fast signals (fluorescence or reflected light)remain fully modulated. Therefore, collecting the im-ages at 0° (in-phase signal plus phosphorescence) and180° (phosphorescence only), using a high-enoughfrequency (e.g., 36 kHz), and finding their differenceallows calculation of the intensity of the in-phasesignal and correction of the intensity images used fordetermination of the phosphorescence-lifetime map.This correction method has been tested with samplescontaining mixtures of a phosphor and a model flu-orophor and shown to effectively remove the in-phasesignals (fluorescence), as long as the latter accountedfor less than 50% of the total intensity.
B. Resolution
For the measurements of the retina of the mouse eye,the imaged area is approximately 0.92 mm high by1.2 mm wide and the CCD array is 480 � 752 pixels.As a result the area per pixel is approximately 1.9 �mhigh � 1.6 �m wide. This area is approximate be-cause no effort has been made to correct forrefractive-index changes along the light path be-tween the coverslip and the retina within the eye.Resolution in the oxygen images is limited by move-ment that occurs during image collection and be-tween images within an image set. There ismechanical coupling between breathing and, to amuch lesser extent, systemic blood-pressure changesand position of the retina and elements within theretina. Thus, although 10 �m vessels can be observedin most individual phosphorescence-intensity im-ages, they can rarely be resolved in the oxygen maps,and then only if the oxygen pressure is substantiallydecreased (intensity increased) relative to that of theneighboring vessels.
C. Phosphor
The phosphor used in this study was polyglutamic Pdtetrabenzoporphyirin dendrimer (Oxyphor G2; Oxy-gen Enterprises, Ltd., Philadelphia, Pa.). Synthesisand properties of this phosphor have been de-scribed.21–23 Oxyphor G2 was injected intravenously as0.15 ml of a 1.6 mg�ml solution in physiological sa-
line. Oxyphor G2 (MW 2642) has 16 carboxyl groupson the periphery, giving a net charge of �16 at phys-iological pH. Because of the large negative charge, ithas a low permeability through the vessel walls. Oxy-phor G2 has absorption maxima at 450 or 635 nm.We chose to excite this phosphor at 450 nm. The bandat 636 nm can also be used, but blue excitation waschosen to minimize the depth to which the excitationlight penetrated into the tissue. The phosphorescence(emission maximum at 810 nm) was measured with a695 nm long-pass Schott glass filter. The values of255 �s and 280 mm Hg s�1 �1 mm Hg � 1 Torr� wereused for the �0 and kQ, respectively,21 at 38 °C.
D. Experimental Protocol
The pigmented mice were anesthetized by intraperi-toneal injection of 0.2 ml of a solution of ketamine�25 mg�ml� and xylazine �25 mg�ml� dissolved inphosphate-buffered saline. A drop of 1% tropicamide(Mydriacyl; Alcon, Ft. Worth, Tex.) was placed on theeyes to dilate the pupils, and Oxyphor G2 (1.6 mg�mlin unbuffered saline, pH 7.5) was given by intrave-nous injection of 0.15 ml into the tail vein. Approxi-mately 4 min after introduction of the Mydriacyl, adrop of hydroxypropyl methylcellulose (Goniosol;CIBAVision Ophthalmics, Atlanta, Ga.) was placedon the eye, and then a small piece of clear plasticsheet was gently placed on the Goniosol. The retinawas then imaged through the coverslip. Phosphores-cence imaging began as early as 3 min after injectionof the phosphor and on occasion continued for asmuch as 1.5 h.
To test the ability of the oxygen maps to detect localvascular abnormalities that generate local hypoxia,pigmented mice were anesthetized, the pupils weredilated with 1% tropicamide, and indocyanine green�5.0 mg�ml 0.25 ml� was injected in the tail vein.Laser photocoagulation was performed with a diodelaser photocoagulator (810 nm; OcuLight Six, IRISMedical, Mountain View, Calif.) and a slit lamp sys-tem with a coverslip as a contact lens. This lasersystem produces vascular occlusion through photo-chemical activation of the indocyanine green withinthe vasculature. The photoinduced radicals causecomplete closure of the vessels in the focal spot of thelaser. Two or three spots of laser photocoagulation90 mW, 75 �m spot size, 0.1 s duration) were appliedto the retina, two to three disk diameters from theoptic nerve of each eye. These spots were far enoughaway from the optic nerve head to avoid hitting majorvessels in the retina and yet still be readily observedthrough the pupil. Two days after laser treatment theOxyphor G2 was injected and the oxygen imaged.This delay was instituted to prevent the local tissueedema and vascular leakage that occur immediatelyafter the treatment. Photocoagulation by use of indo-cyanine green as an intravascular sensitizer was cho-sen to alter the local retinal blood supply while itminimized direct tissue injury.
3. Results
A. Imaging Phosphorescence Intensity from theVasculature of the Retina
The camera was focused on the retina, the brightnessadjusted, and a sequence of images collected. Thesequence of images used delays ranging from 0° to360° relative to the excitation. A sequence of phos-phorescence intensity images, collected by use of1030 Hz modulation, is shown in Fig. 1. There is anetwork of small vessels between the major arteriolesand veins in the retina that appear to be quite uni-formly �10 �m in diameter. We refer to this networkof small vessels as capillaries, although this is likelyto be an oversimplification. Observing these smallvessels requires that there be little movement of themouse during acquisition of the image.
As shown in Fig. 1, the individual images of phos-phorescence intensity show with good resolution theveins and the arteries that supply blood to the retina.The images are similar to those obtained during flu-orescein angiography. In both cases, the luminophorsare dissolved in the blood plasma and, therefore, theintensity images show vascular structure. Leaks ofeither fluorescein or the phosphor through the vesselwalls lead to the appearance of small local regions ofincreased intensity spots in the images, and both theintensity and the size increase with time after phos-phor injection. The permeability of fluorescein to thevessel walls is much higher than that of Oxyphor G2(charge, 1 compared with �16; molecular weight, 332compared with 2,642). The change in the image thatis due to dye leakage is greater for the phosphor thanfor fluorescein. As phosphor leaks out of the vessel,the phosphorescence intensity increases not only be-cause of the increase in the amount of dye near thelocus of the leak but also because the oxygen pressureis lower in the extravascular space than in the ves-sels.
The phosphor is present only in the blood plasma,so the phosphorescence intensity in the images isrelated to both the blood volume and the oxygen pres-sures. Thus the lower oxygen pressures in the veinsrelative to the arterioles result in the veins’ beingbrighter than the arterioles. Larger vessels arebrighter than small vessels, and the capillary bedareas, which lack vessels larger than �10 �m diam-eter, show much weaker phosphorescence than thelarger vessels. Individual capillaries can be seen inthe phosphorescence images but are not resolved inthe lifetime and oxygen maps unless they are blockedor otherwise depleted in oxygen relative to the sur-rounding capillaries.
B. Maps of Phosphorescence Lifetime andOxygen Pressure
Typically, a set of phosphorescence intensity images(see above) with phase delays of 0° to 360° and at 30°intervals was taken. The in-phase correction was ob-tained by imaging with frequency 36 kHz at 0° and180° (see above). Subtracting the 180° image from the0° image yields the intensity image that corresponds
to lifetimes of less than 1 �s, i.e., reflected light andfluorescence. This in-phase signal was subtractedfrom all images in the set to give a set of correctedimages for calculation of the phosphorescence life-times. Figure 2 shows a plot of the phosphorescenceintensity in three regions of interest, corresponding
to two veins and an arteriole, as a function of thephase delay used to collect the intensity images. Thesets were fitted to sinusoids in each pixel, and thephase shift was determined for each fit. Thephosphorescence-lifetime image was calculated fromEq. (3) and converted to the oxygen image by use ofEq. (8).
The phase delay image, the phosphorescence-lifetime image and, the oxygen pressure image aredisplayed in Fig. 3. Visualizing the phase delay imageis important because the conversion of the lifetimeimage into the oxygen image can be done most reli-ably if the lifetimes are determined at frequencies atwhich the delays are 25° to 35°. Each phosphor ischaracterized by a distribution of lifetimes and,therefore, changing the phase delay in a frequency-domain measurement can lead to differences in theapparent lifetimes, �. The narrower the distributionin lifetimes, the less effect the difference in phasedelays has on the calculated lifetime. When therewere regions in the map with phase shifts that dif-fered substantially ��10°� from the phase �28°� usedfor phosphor calibration, they were imaged again at afrequency for which the phase shift was nearer 28°.
In the region of the optic nerve’s head, the imagesshow relatively large veins and arterioles radiatingfrom a small central region (Fig. 3). As you progressaround the disk, the vessels alternate between arte-
Fig. 2. Phosphorescence intensity versus phase delay (in degrees)for three regions of interest in the image, two veins and one arteriole.Least-squares fits to a sinusoid yielded phase shifts of 43.6° and40.4° for the veins and 35.1° for the arteriole. (Graphed in Origin.)
Fig. 3. Maps of (a) the phosphorescence intensity at 30° phase shift, (b) the phase shift between excitation and phosphorescence, (c) thephosphorescence lifetime, and (d) the oxygen pressure calculated from the set of images in Fig. 1. The maps correspond to a region whosephysical dimensions are 0.92 mm high by 1.2 mm wide. The oxygen maps were smoothed with a running-median 5 � 5 filter.
rioles and veins, and this is clearly seen in both life-time and oxygen images. The vessels alternatebetween low and high oxygen pressures and long andshort phosphorescence lifetimes. The oxygen pres-sure in the veins is typically 30–45 mm Hg, whereasthat in the arterioles is 60–80 mm Hg. In regionsfarther from the optic nerve’s head there are substan-tial areas of capillary bed that exhibit higher oxygenpressures, usually 50–100 mm Hg. This result is con-sistent with some of the blue �450 nm� excitationlight passing through the retina and exciting a smallamount of diffuse phosphorescence from the under-lying choriocapillaris where the oxygen pressure ishigh (short lifetime). When this choroidal signal isadded to the weak phosphorescence from the retinalcapillaries, the resultant mixed signal gives calcu-lated oxygen pressures between those of the retinaand of the choroid. The total intensity is very low,such that the oxygen values also become noisy.Where the phosphorescence signal from the retinalvessels is higher, such as for the veins and arterioles,the oxygen pressures in the individual vessels can beaccurately determined.
C. Dependence of the Retinal Oxygen Maps on Timeafter Injection of the Anesthetic
When mice are given anesthetics, the animals oftengo through a transient period of decreased blood
pressure, abnormal breathing patterns, or both.The duration and severity of this period are quitevariable among animals owing to individual differ-ences in biochemical and physiological responses tothe anesthetic. In the experiment shown in Figs. 4and 5, oxygen maps were repetitively measured at1–2 min intervals from the earliest time attainable�3 min� until the mouse began to recover from anes-thesia. Representative data are shown in Fig. 4 as thephosphorescence intensity images and oxygen pres-sure maps at 5 and 35 min after anesthesia, shortlybefore the mouse awakened. In this animal, the ve-nous oxygen pressures increased with time after an-esthesia from �15 mm Hg at 5 min to �50 mm Hg at35 min. In contrast, the arteriolar values were essen-tially constant and near 75 mm Hg. The time courseof the process is shown in Fig. 5, where the oxy-gen pressures in selected regions of two arteriolesand two veins are plotted against the time afteranesthesia.
The time course of the oxygen measurements afterinduction of anesthesia is highly variable amongmice, ranging from almost no change over time tofailure to attain stable levels before recovery (usuallybecause of an erratic breathing pattern). In mostcases, however, there is a transient lowering of theoxygen pressure in the veins, which returns to a
Fig. 4. Dependence of the retinal oxygen pressures on time after induction of anesthesia. The mouse was given an intraperitonealinjection of anesthetic, the pupil was dilated, and retinal imaging of the phosphorescence lifetimes began �3 min later. The measurementsshown were made at 5 and 35 min after induction of anesthesia. The area of the retina that was imaged was approximately 0.92 mm highby 1.2 mm wide.
nearly stable condition within 5–10 min. In theperiod when the values are stable, the arteriolarvalues are 65–85 mm Hg and the venous values35–55 mm Hg. Oxygen extraction is 25–30 mm Hg.Similar time dependence has been observed formicrovascular oxygen levels in normal muscletissue in mice used as controls for tumor oxygenmeasurements.24
D. Identifying Vascular Lesions Induced by Alterations inTissue Oxygenation
In initial experiments, a laser was used to block oneof the larger retinal blood vessels and the phospho-rescence images were collected 48 h later. The mea-sured phosphorescence was well defined, with no
evidence of leakage from the vessels (spreading ofintensity, increase in intensity with time, or both).The phosphorescence from the large damaged areawas high owing to the low oxygen levels, and thisobscured the emission from the surrounding normaltissue. As a result, the oxygen maps showed onlylarge areas with oxygen pressures near zero, confirm-ing the presence of a massive injury. This model wasnot further pursued because such large injuries canbe readily detected by simple ophthalmoscopy. It wasmore interesting to test for the ability to detect injurythat is due to the pathology involving microvesselfailure, where blockage of capillaries would resultonly in small hypoxic regions. To this end, a model oflocal vascular failure was generated. Two or three75 �m focal spots of laser photocoagulation with in-docyanine green as an intravascular sensitizer weremade in regions that spared observable major bloodvessels. This procedure restricted injury to the capil-lary bed within the focal area. Oxygen measurementswere made 48 h later to limit the contribution of im-mediate local edema and vascular leakage. An oxy-gen pressure map of a region with two laser-inducedlesions is shown in Fig. 6.
The lesions appear in phosphorescence intensityimages as bright spots (not shown). In the oxygenmap they appear as nearly round areas with centralcore oxygen values well below those of the surround-ing tissue. The absence of significant leakage of thephosphor from the vessels was confirmed by mea-surements made over periods of 1 h following phos-phor injection, during which time the size and theoxygen profile of the region of hypoxia remained con-stant. Figure 6 presents an oxygen map of one of thetwo lesions reimaged at a lower frequency �800 Hz� toyield a better estimate of the core oxygen pressure.The laser-induced lesion consists of a central regionof acute hypoxia surrounded by a region of gradedoxygen deficit that extends outward to approximately150–200 �m from the center. The mean oxygen pres-sure in the core was less than 7 mm Hg.
Fig. 5. Time course of the oxygen pressures in the retinal veinsand arterioles following anesthesia. Oxygen pressure maps of theretina were repetitively measured; a total of 19 measurements wasmade over the period of 3–35 minutes following anesthesia. Re-gions of interest were selected within two arterioles and two veins,and the average oxygen pressures for each region were determinedat every time. The resultant values are plotted as a function of timeafter anesthesia.
Fig. 6. Oxygen pressure maps of (a) the region containing two laser photocoagulation spots and (b) a region centered on one of the twospots. The measurements were made with modulation frequencies of 3030 and 800 Hz, respectively. The area of retina covered by theoxygen maps is approximately 0.92 mm high by 1.2 mm wide.
Images of the retina in mice, particularly old obesemice, occasionally show anomalous hypoxic vesselswithin the capillary bed of the retina. An example ofsuch vessels is shown in Fig. 7. The phosphorescenceintensity image [Fig. 7(a)] does not show any obviousvascular anomalies, whereas when the oxygen pres-sure map is calculated there are vessels in three re-gions with retinal capillaries that resemble, on amicroscale, varicose veins. The oxygen pressures inthese vessels are well below that in the surroundingcapillaries. It is likely that they have greatly dimin-ished blood flow and that, as a result, much moreoxygen has been extracted from the blood withinthese vessels.
4. Discussion
Oxygen measurements by quenching of phosphores-cence originating from phosphors dissolved in theblood plasma in vivo can be compared with tissueoxygen measurements made with micro-oxygen elec-trodes. This has been done for the cat eye (see Shonatet al.14 for comparison with Linsenmeier2), for skele-tal muscle,25 and for tumors growing in windowchambers.26 In each case, oxygen electrodes were in-serted into the tissue, whereas the phosphor was inthe blood serum. The values measured by the oxygenelectrodes are generally slightly lower than thosemeasured by phosphorescence lifetime. The differ-ence indicated that there is a small decrease in theoxygen pressure from the microcirculation to the ex-tracellular space, a decrease consistent with that pre-dicted by oxygen diffusion. Several investigators,including Linsenmeier and co-workers2,3 and Yu etal.,27–29 have used oxygen-sensitive microelectrodesto measure oxygen tension in the retina. These stud-ies have concluded that tissue hypoxia may contrib-ute to the development of pathology in the retina of adiabetic animal but not in the Royal College of Sur-geons model for retinitis pigmentosa. The data, how-ever, apply only to models of global hypoxia, in which
all or at least a large fraction of the tissue becomeshypoxic. In most diseases of the eye, however, thepathology is likely to involve functional failure at thelevel of individual capillaries. This would initiallyresult in localized regions of focal hypoxia, but as thenumbers of defective vessels within a given regionaccumulated the region with an oxygen deficit wouldincrease. Oxygen electrodes make point measure-ments and are statistically unlikely to hit a region offocal hypoxia. If one were hit, slight movement of theelectrode tip would lead to a widely different value,and the measurement would likely be considered anartifact. Thus oxygen electrodes can effectively mea-sure oxygen in macroscopic areas but are less able todetect or evaluate small regions of hypoxia inter-spersed among much larger volumes of normoxia. Asa result, they are not suitable for evaluating earlystages of pathologies in which the failures occur inindividual capillaries and therefore cause only smallfocal regions of tissue hypoxia.
Phosphorescence-lifetime imaging is suited to eval-uation of microcirculatory functions for the followingreasons: The measurements are noninvasive, i.e., donot involve physically entering the eye or inserting anobject into the retina; repetitive measurements canbe made in the same animal over time; and the oxy-gen maps have excellent spatial resolution and caneven identify and quantify vascular failure at thelevel of individual retinal capillaries.
Oxygen-dependent quenching of phosphorescencehas been shown to produce good maps of the distri-bution of oxygen in the retina of the mouse eye. Thisopens a wide range of applications to experimentalmodels of diseases of the eye, many of which are inmice and rats. It is not yet possible to discuss withcertainty the concentration of oxygen in normal ret-inal tissue because it is necessary to immobilize theanimals with anesthetics. Anesthetics can alter thecardiopulmonary function, changing blood pressure,cardiac output, breathing rate, etc., with subsequentincreases or decreases in tissue oxygenation. The
Fig. 7. Phosphorescence intensity image (left) and oxygen pressure map (right) of the eye of a 2-year-old mouse. The measurements weremade at 1000 Hz. The retinal area imaged was approximately 0.92 mm high by 1.2 mm wide.
measurements are selective for the retina, as the cho-roidal vasculature lies behind the highly absorbingand scattering pigmented layer. The choroidal vascu-lar bed, therefore, contributes only a weak, highlyscattered background phosphorescence with a shortlifetime. Although emission from the choroid can in-terfere with the oxygen pressure measurements inthe retinal capillaries during normoxic conditions,hypoxic retinal capillaries have brighter phosphores-cence and can be readily observed and the oxygenlevel estimated.
5. Summary
The phosphorescence-lifetime imaging method is auseful tool for studying eye physiology and pathology.It has been shown to be able to identify and charac-terize vascular anomalies at the level of individualretinal capillaries. As a result, phosphorescencequenching is well suited for critical evaluation of thehypothesized role of local hypoxia in diseases such asdiabetic retinopathy and age-related macular degen-eration. Where as local hypoxia has a causative rolein, or accompanies, early development of disease,such measurements can contribute significantly toboth early diagnosis of the disease and monitoringprogression of the pathology. Oxygen measurementsmay also provide a basis for selecting pharmaceuticalagents that delay or prevent the development of tis-sue hypoxia and thereby delay or prevent loss ofvision.
Disclosures
D. F. Wilson and S. A. Vinogradov hold several pat-ents on technology related to phosphorescence mea-surements of oxygen. Some of these patents havebeen licensed to Oxygen Enterprises, Ltd., a companyin which D. F. Wilson has significant holdings. Noneof this research was sponsored by a commercialentity.
This research was supported in part by grants NS-31465, HD041484, and R43-DK064543 from the U.S.National Institutes of Health.
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