6
A Method of Photographing Fluorescence in Circulating Blood in the Human Retina By HAROLD R. NOVOTNY, B.S., AND DAVID L. ALVIS, M.D. T HE PHYSIOPATHOLOGY of the reti- nal vasculature would be better under- stood if more were known about blood flow in these vessels. Because of the unique quality of transparency in the eye, methods depend- ing on direct observation of the retinal ves- sels seem especially inviting. Already re- ported by various authors are technics for measuring the changes in caliber of retinal vessels,1 and methods of observing retinal blood flow by cinematography,2 and, in cats, by injecting trypan blue. Although useful, these methods have certain limitations, and additional means of observing retinal blood flow with increased visibility and definition are needed. The purpose of this paper is to describe a method for the study of retinal blood flow in man by the use of intravascular fluorescein and retinal photography, and to report some preliminary observations made with this method. Materials and Methods The Zeiss fundus camera was used. It was equipped with an electronic flash, the maximum discharge of which allowed one photograph every 12 seconds. Light intensity of the electronic flash was set at position III, and retinal photographs were made of the luminescence of fluorescein as it passed through the retinal vessels. In order to do this, both activating and emitting wave lengths of blood-fluorescein mixtures were determined spectrofluorometrically. The optimal activating wave length was found to be 490 mju, in the blue range of the visible spectrum; and the maximal From the Departments of Medicine and Ophthal- mology, Indiana University School of Medicine, Indianapolis, Indiana. Supported in part by contract AF 41 (657-225) monitored by the School of Aviation Medicine, Brooks Air Force Base, Texas, in part by grants from the Life Insurance Medigal Research Fund and the Knights Templar Eye Foundation, and in part by grant 2B5034, U. S. Public Health Service. emitting wave length was 520 m[i, in the green. Kodak wratten filters no. 47 and no. 58, combined with a 3-nmm. layer of 0.25 M copper sulfate, were accordingly inserted into the optical system (figs. 1 and 2) at appropriate points. In order to modify the activating light, the blue no.-47 filter was placed in the path of the beam from the electronic flash and from the incandescent viewing source. This made it possible to see, as well as to photograph, the fluorescence as it developed and faded. The green no.-58 filter with the copper sulfate layer was placed in the path of emitted light in order to absorb background illumination and increase the contrast between it and the trans- mitted fluorescence. The green no.-58 filter per- mitted approximately 5 per cent transmittance in the blue range, which gave a visible background in the retinal photograph irrespective of the fluorescence. The layer of copper sulfate solution was not essential, but slightly sharper negatives were obtained with it. Ansco Super Hypan 35-nim. film was used; it was force developed for 10 minutes at 70 F. with UFG Ethol developer, placed in an acetic acid stop bath, and fixed for 10 minutes with Kodak acid fixer. Prints were made on Kodak Medalist F-2 or F-4 photographic paper, depending on the contrast desired. In each patient, a control picture of the fundus was taken prior to injection of fluorescein. Then, in a darkened room, 5 ml. of Fluoreseite* were rapidly injected into an antecubital vein and were followed by 5 ml. of normal saline to deliver a con- centrated bolus of dye into the circulation. The first photograph was made when arterial fluo- rescence appeared, followed by serial photographs at 12-second intervals for approximately 31/2 minutes. Results Normal Patients The time from injection into the antecubital vein until visualization of fluorescence varied from 12 to 30 seconds, when a striking lumi- *Fluorescite is 5 per cent fluorescein in sodium bicarbonate, an injectable product of the C. F. Kirk Company, 521 W. 23rd St., New York 11, New York. Circulation, Volume XXIV, July 1961 82 by guest on October 4, 2017 http://circ.ahajournals.org/ Downloaded from

AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

  • Upload
    others

  • View
    55

  • Download
    0

Embed Size (px)

Citation preview

Page 1: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

A Method of Photographing Fluorescence in CirculatingBlood in the Human Retina

By HAROLD R. NOVOTNY, B.S., AND DAVID L. ALVIS, M.D.

T HE PHYSIOPATHOLOGY of the reti-nal vasculature would be better under-

stood if more were known about blood flowin these vessels. Because of the unique qualityof transparency in the eye, methods depend-ing on direct observation of the retinal ves-sels seem especially inviting. Already re-ported by various authors are technics formeasuring the changes in caliber of retinalvessels,1 and methods of observing retinalblood flow by cinematography,2 and, in cats,by injecting trypan blue. Although useful,these methods have certain limitations, andadditional means of observing retinal bloodflow with increased visibility and definitionare needed.The purpose of this paper is to describe

a method for the study of retinal blood flowin man by the use of intravascular fluoresceinand retinal photography, and to report somepreliminary observations made with thismethod.

Materials and MethodsThe Zeiss fundus camera was used. It was

equipped with an electronic flash, the maximumdischarge of which allowed one photograph every12 seconds. Light intensity of the electronic flashwas set at position III, and retinal photographswere made of the luminescence of fluorescein asit passed through the retinal vessels. In order todo this, both activating and emitting wave lengthsof blood-fluorescein mixtures were determinedspectrofluorometrically. The optimal activatingwave length was found to be 490 mju, in the bluerange of the visible spectrum; and the maximal

From the Departments of Medicine and Ophthal-mology, Indiana University School of Medicine,Indianapolis, Indiana.

Supported in part by contract AF 41 (657-225)monitored by the School of Aviation Medicine,Brooks Air Force Base, Texas, in part by grantsfrom the Life Insurance Medigal Research Fund andthe Knights Templar Eye Foundation, and in partby grant 2B5034, U. S. Public Health Service.

emitting wave length was 520 m[i, in the green.Kodak wratten filters no. 47 and no. 58, combinedwith a 3-nmm. layer of 0.25 M copper sulfate, wereaccordingly inserted into the optical system (figs.1 and 2) at appropriate points.

In order to modify the activating light, theblue no.-47 filter was placed in the path of thebeam from the electronic flash and from theincandescent viewing source. This made it possibleto see, as well as to photograph, the fluorescenceas it developed and faded.The green no.-58 filter with the copper sulfate

layer was placed in the path of emitted light inorder to absorb background illumination andincrease the contrast between it and the trans-mitted fluorescence. The green no.-58 filter per-mitted approximately 5 per cent transmittance inthe blue range, which gave a visible backgroundin the retinal photograph irrespective of thefluorescence. The layer of copper sulfate solutionwas not essential, but slightly sharper negativeswere obtained with it.Ansco Super Hypan 35-nim. film was used; it

was force developed for 10 minutes at 70 F. withUFG Ethol developer, placed in an acetic acidstop bath, and fixed for 10 minutes with Kodakacid fixer. Prints were made on Kodak MedalistF-2 or F-4 photographic paper, depending on thecontrast desired.

In each patient, a control picture of the funduswas taken prior to injection of fluorescein. Then,in a darkened room, 5 ml. of Fluoreseite* wererapidly injected into an antecubital vein and werefollowed by 5 ml. of normal saline to deliver a con-centrated bolus of dye into the circulation. Thefirst photograph was made when arterial fluo-rescence appeared, followed by serial photographsat 12-second intervals for approximately 31/2minutes.

ResultsNormal Patients

The time from injection into the antecubitalvein until visualization of fluorescence variedfrom 12 to 30 seconds, when a striking lumi-

*Fluorescite is 5 per cent fluorescein in sodiumbicarbonate, an injectable product of the C. F. KirkCompany, 521 W. 23rd St., New York 11, New York.

Circulation, Volume XXIV, July 196182

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from

Page 2: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

FLUORESCENCE IN HUMAN RETINA

70

60X #47 Kodak Wratten Filter

Z 50s-o'S | \ ] #58 Kodak Wratten Filter1-

30 / 0 { \1with 3 mm layer of Cu40 lyrC O

z 30

1- 20

10-

0300 350 400 450 500 550 600 650 700 750 800

WAVE LENGTH IN MILLIMICRONS

Percentagecamera.

Figure 1transmittance of filters;used in the

nescenee appeared in the retinal vessels asthe fluorescein passed through them.

Separate arteriolar and venous filling phaseswere present in the serial photographs (figs.3 and 4). During and after the venous fillingphase, a generalized background mottlingdeveloped, presumably representing the cho-roidal circulation. Near the end of the 31/2minute period of photography, contrast fadedand the retinal vessels were difficult to see.

There were considerable differences in thecirculation times from arteriolar to venoussides in different portions of the retina, thearteriovenous transit time being fastest inthe region of the fovea, and slowest in theperipheral portions of the retina. An occa-sional arteriolar twig emptied more slowlythan the others of similar size at comparabledistances from the disk. One arteriolovenousshunt was seen.

Stratified flow occurred in many of thelarger vessels. In the larger arterioles theflow rate seemed more rapid in the centralportion of the vessel than along the walls,since fluorescence appeared first and clearedfirst in the central portions. In the largerveins there was often a reversal of this pat-tern, the lateral portions of the venous streamfirst showing fluorescence, sometimes on onlyone side of the vein, as fluorescent bloodentered the vein from small branches nearthe disk. These fluorescent streams tendedto maintain their lateral position along thesame side of the vein all the way to the

Circulation, Volume XXIV, July 1961

Figure 2Cross section of optical system of Zeiss funduscamera with filters inserted.

disk. Two or three minutes after the injection,some larger veins continued to show moreprominent residual fluorescence along thewalls than in the central portions. It was notclear whether this represented a fluorescentplasma cuff or fluorescence of the vessel wallitself.Hypertensive and Diabetic Patients

In addition to the same findings as in thenormal eye, fluorescence in some hypertensiveand diabetic patients showed smaller, more

obscure vascular patterns that were difficultor impossible to see with the ophthalmoscopeor in ordinary retinal photographs. New ves-

sel formation was one of the most strikingfindings in these patients. Fluorescence ap-peared in microaneurysms, vasoproliferativeareas, and tortuous small vessels as well as

in the normal circulation (figs. 5 and 6).Fluorescence could not be seen in hemorrhagicareas, although it appeared early in some

cotton-wool patches and remained throughoutthe period of photography. Some patches andedema residues failed to become fluorescent.

DiscussionSerial fluorescence-photography of the hu-

man retinal vasculature provides a dynamicrecord and increased visibility of the vascularpattern and blood flow by means of a simpletechnic.

83

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from

Page 3: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

NOVOTNY, ALVIS

Figure 3 Figure 4Normal patient. Top. The arteriolar filling phase. Normal patient. Top. Arteriolar filling phase withBottom. the venous filling phase. Arrows on the laminar flow in the veins. Bottom. The venousleft indicate differential emptying of comparable- filling phase.sized arterioles, and the arrow on the right indi-_ - -1__- -1 7_ _I-- _-- _ 7cates the rapid passage of the aye from an arterioleto a venule through a small vessel resembling anarterioloivenous shunt.

Activation by ultra-violet light was foundto be unnecessary; filtered visible lightavoided the potential hazards of ultra-violetexposure, and the natural fluorescence of thedens was no problem. The eventual appearanceof fluorescein in the aqueous humor, however,contributed to the loss of definition in laterserial photographs.The background mottling, presumably from

the choroidal circulation, which persistedthroughout the period of photography, sug-gested that the rate of fluorescein turnoverin the choroid may be slower than that oft1e retina.

*At prevent, owing to the limitations of theelectronic flash apparatus, pictures cannotbe taken more often than every 12 seonds.

A new incandescent light source and a rapidfilm-changer may eventually permit exposureto be made in more rapid succession. Also,retinal cinematography2 would seem particu-larly adaptable to this method.Further studies with the technics described

here are being carried out to determine therate of change of optical density of veinsin the photographic negatives, to determinewhether a relationship can be establishedbetween dye concentration and image densityof the vessel. While such a relationship can-not give an estimate of retinal flow in absoluteterms, it could provide a ratio between flowin control and experimental states, or betweenflow rates in different veins of the same eye.

SummaryA simple method, with use of intravenous

fluorescein, was used for producing and photo-Circultion, Volume XXIV. Ju1y 1961

84

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from

Page 4: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

FLUORESCENCE IN HUMAN RETINA

Figure 5Jfq1pe r te)isive patienit. Top. Mawde with KodaklIPlas-X film befor.e the injection of fiinor'es(celnl.Middle. Madlc ni/Super Hilpan flim and showsthe 1i'i)iois filling,9 phase. Bottom. T'aken 3112m tin/les (ijter injection. Thfi a'rr()' neartftie opticn~ert1ie heead (middle figure) points to a cottonw'ool exu/date that is b((o)niing fluorescent (1)1d thatletiell)os marked flunresrence in the bottom figure.The second a r rowv (middle figure) indicates aregion th~at shows only a fewv scattered hemorrhagesi.n the )ion fluorescenit p),icturee (top figure) but that(levelops dlefiniite fluoresceence (inmidclle anld bottomfigures), suggesting that capillaries throughout theret'giontmug1C UP(1h )ve lrmormIll/ great permisieabilitly.Seeral u/hetr( regin. oa increased permeability aree cidlent.

Circulation, Volumne XXIV, July 1961

Figure 6Diabetic patient. Top. Made Itith Kolak Plus-Xfilm be for the injection of flutorscein. Mliddle.The arteriqla r fillinp/l uase. The a rro,wpoints toa region of udo casca la riiation and increased capil-lary permeability. Bottom. Venit)us filling. Thetirrawes more (distant to the dlish' indicates threenteicroanergsms that htare became fluor escent.

graphing fluorescence ill (irculatinmug l)]ood ofthe humian retina.

Separa-te arteriolar and venions fillillgplases, 811arten i luvemous slhunt, sluggishchoroidal circulation, stratifie(d flow of fluo-

85

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from

Page 5: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

NOVOTNY, ALVIS

rescein, and rapid central retinal circulationtimes were observed in normal retinas.

Similar findings were seen in hypertensiveand diabetic patients, and, in addition, neo-vascularization was clearly defined, and somecotton-wool patches, but not hemorrhages,were found to fluoresce.

Limitations and applications of the methodare discussed.

AcknowledgmentGrateful appreciation is extended to John B.

Hickam, M. D., and Fred M. Wilson, M. D., of theDepartments of Medicine and Ophthalmology, respec-tively, for their assistance and criticism. The work

of James Hartigan and others at Eli Lilly Companyin determining spectrofluorometric data was mosthelpful.

References1. CUSICK, P. L., BENSON, 0. 0., JR., AND BOOTHBY,

W. M.: Effect of anoxia and of high concen-trations of oxygen on the retinal vessels:Preliminary report. Proe. Staff Meet., MayoClin. 15: 500, 1940.

2. BAILEY, P., JR., AND SWAN, K. C.: Cinematog-raphy of human retinal vessels. J.A.M.A.170: 1373, 1959.

3. CHAO, P., AND FLOCKS, M.: The retinal circula-tion time. Am. J. Ophthalmol. 46: 8, 1958.

THIRD OBSERVATIONOf the Preternatural or Morbid Sound of the Chest and Its General Import

X. To be able justly to appreciate the value of the various sounds elicited from thechest in cases of disease it is necessary to have learned, by experience on many subjects,the modifications of sound, general or partial, produced by the habit of the body, naturalconfirmation as to the scapulae, mammae, the heart, the capacity of the thorax, thedegree of fleshiness, fatness, etc., inasmuch as these various circumstances modify thesound very considerably.-From On Percussion of the Chest. Published in 1761. Trans-lated by John Forbes, M.D. In: Classics of Medicine and Surgery. New York, DoverPublications, Inc., 1959, p. 127.

Circulation, Volume XXIV, July 1961

86

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from

Page 6: AMethod of Photographing Fluorescence Circulating Blood in ...AMethodof Photographing Fluorescence in Circulating Blood in the HumanRetina By HAROLD R. NOVOTNY, B.S., AND DAVID L

HAROLD R. NOVOTNY and DAVID L. ALVISRetina

A Method of Photographing Fluorescence in Circulating Blood in the Human

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 1961 American Heart Association, Inc. All rights reserved.

75231is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TXCirculation

doi: 10.1161/01.CIR.24.1.821961;24:82-86Circulation. 

http://circ.ahajournals.org/content/24/1/82located on the World Wide Web at:

The online version of this article, along with updated information and services, is

  http://circ.ahajournals.org//subscriptions/

is online at: Circulation Information about subscribing to Subscriptions: 

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

  document. Permissions and Rights Question and Answer

of the Web page under Services. Further information about this process is available in thewhich permission is being requested is located, click Request Permissions in the middle columnClearance Center, not the Editorial Office. Once the online version of the published article for

can be obtained via RightsLink, a service of the CopyrightCirculationoriginally published in Requests for permissions to reproduce figures, tables, or portions of articlesPermissions:

by guest on October 4, 2017

http://circ.ahajournals.org/D

ownloaded from