Two-point fluorophotometer for the human ocular fundus

Charles Riva and Isaac Ben-Sira

A new technique for making on-line recordings of fluorescein dilution curves from two locations in the

human retina has been devised. A modified Zeiss fundus camera with two optical fibers mounted in a

scanning ocular collects the light emitted by fluorescein dye from two spots in the fundus. Two fiber-optic

cables transmit the collected light to a photomultiplier. The detector output current is transmitted to a

two-channel pulse sorter. The output signals from the pulse sorter are amplified and displayed on a two-

channel recorder. Since this technique utilizes only small amounts of fluorescein, multiple measurements

in the same subject are possible.

1. IntroductionDetermination of the blood flow rate in the retina

is fundamental to the understanding of basic physio-logical processes and pathological conditions in the

ocular fundus. Flow measurements are also essentialfor the objective evaluation of treatment of oculardiseases. Currently, the blood flow in a portion of

the retina supplied by one artery, the so-called seg-mental retinal blood flow (SRBF), is estimated by

means of the fluorescein dilution techniquesIn this technique, fluorescein is injected into the

circulatory system, and a dilution curve for the dye,i.e., the time course of its relative concentration, is

recorded from a retinal artery (inflow of the segment)and from a corresponding retinal vein (outflow of thesegment). From the recordings of the arterial andvenous dilution curves, the mean circulation time of

the dye (MCT) through the retinal segment can be

determined. This segment is assumed to constitutea closed system, that is, there is no loss of dye be-tween the inflow and the outflow. After estimationof the volume of blood (V) in such a segment, the

SRBF can be obtained by using the relation SRBF =

V/MCT.The conventional technique for obtaining dilution

curves requires densitometric measurements of agreat number of fundus fluorescence photographs.This photographic method is laborious and time con-

suming and thus has never been applied on a routineclinical basis.

The authors are with the Department of Retina Research, Eye

Research Institute of Retina Foundation, Boston, Massachusetts02114.

Received 3 June 1975.

Therefore, we have developed a simple methodthat permits on-line recordings of dilution curvesfrom an artery and a vein simultaneously, or fromany other two locations in the retina. Our methodemploys a standard Zeiss fundus camera (Fig. 1),which was modified as follows (Fig. 2).

II. Illumination of the Fundus

The Xenon flashbulb Ls was replaced by a 100-Whalogen lamp L (Sylvania FDX), which is connectedto a current-regulated power supply. The semitrans-parent mirror P was removed, because the illumina-tion source W is not used. In the plane (a,a), conju-gated to the retina of the subject, a diaphragm D hasbeen mounted. The diameter -of its aperture is soadjusted that the field of retinal illumination barelyencompasses the two sites of measurement. Thus,only a small retinal field is illuminated, resulting in areduction of the amount of fluorescent light originat-ing from regions of the fundus other than the record-ing sites. The fundus is illuminated in the range ofthe excitation spectrum of fluorescein by inserting afilter Fex (Spectro-tech SE 40). The spectral charac-teristics of Fex are shown in Fig. 3. In this system,retinal irradiance is equal to or less than 1.3 X 10-3

W/cm2 , which is at least 70 times less than the maxi-mum permissible retinal irradiance for continuoussources.2

Ill. Stabilization of the Patient's Eye

Meaningful recordings of dilution curves from asmall spot in the fundus can be obtained only if theexamined eye remains sufficiently stable. As dis-cussed elsewhere, 3 this can be achieved by having theexamined eye fixate on a punctual target. To per-form measurements of dilution curves from sites nearthe optic nerve, this fixation target, or an image of it,must be located within the solid angle centered at the

November 1975 / Vol. 14, No. 11 / APPLIED OPTICS 2691

Fig. 1. Schematic diagram of unmodified Zeiss fundus camera:LS = xenon flashbulb; P = semitransparent mirror; W = doublefilament bulb; OL = ophthalmoscopic lens; R = eyepiece reticule;C = camera housing; F = plane of photographic film. Rays shownrepresent light incident on the eye (--) and reflected from the eye

(-).

Fex

Fig. 2. Schematic diagram of modified Zeiss fundus camera: L =100-W halogen lamp; D = diaphragm; F = excitation filter forfluorescein; SM = glass plate; F,F 2 = optical fibers. The part ofthe figure to the right of the arrow has been magnified. The

planes (x,x) and (y,y) are conjugated to the plane of the retina.

pupil of the subject that extends to the periphery ofthe ophthalmoscopic lens OL. This has beenachieved by mounting a fixation target in the plane(y,y), the plane of the eyepiece reticule R in the stan-dard Zeiss eyepiece. This plane is conjugated to thepatient's retina. Our target is the aperture of a smallfiber (200 gm in diameter) that can be moved in theplane (y,y) by means of two microdrives. The otherextremity of the fiber is illuminated by a 0.5-mWHe-Ne laser beam.

The subject views the target through the semi-transparent mirror SM (4% reflection) and the col-lecting optics of the fundus camera. For measuringmore peripheral areas of the retina, we use a Haag-Streit slit-lamp fixation system (part 6604), in whichthe standard fixation pattern has been replaced by apinhole 25 m in diameter.

IV. Detection of FluorescenceIn the plane (x,x), conjugated to the retina (the

film plane), two optical fibers, F, F 2, mounted in a

scanning ocular, collect the light emitted from twospots in the fundus. The diameter of their input ap-erture is 450,um. F is fixed slightly off center. F 2can be moved along a straight line in the plane (x,x).The whole ocular scanning system can be rotatedaround the axis of the observation system of the cam-era. Any pair of locations in the fundus within thefield of the Zeiss fundus camera can be reached byusing the target light, rotating the scanning ocular,and moving F 2. In the plane (x,x), the fundus is im-aged with a magnification of 2-3X. As a result, thecollecting aperture of the fibers is only slightly largerthan the diameter of the main arteries and veins nearthe optic disk.

The light collected by these fibers is transmitted tothe cathode of a photomultiplier (EMI 9558) by twofiber-optic cables. A barrier filter (Spectro-tech SB50) has been mounted in front of the photomultiplier.Its spectral transmission is shown in Fig. 3. A diskwith two apertures rotates in front of the detector, al-lowing alternate illumination of the photocathode byeach filter.

A two-channel pulse sorter separates the electricalsignals associated with the light signals from eachfiber. After amplification and electronic filtering,the output signals, which are proportional to the flu-orescence intensity, are displayed on a two-channelrecorder.

The sensitivity of the whole system is such thatgood fluorescein curves can be obtained in youngsubjects with only 10 mg of fluorescein, compared tothe 200-500 mg of fluorescein used for photographicdetection. Multiple injections of fluorescein aretherefore possible. Before each successive injection,the output signal is brought back to the baseline byusing a track and hold system similar to that de-scribed previously. 4

V. Representative MeasurementsFigure 4 shows typical dilution curves recorded

from an artery and a vein in the superior temporal

100

EXCITAr/MN80-

. 607

R 40t

_A

Wavelength (nm)

Fig. 3. Spectral transmission characteristics of the excitation andemission filters used to detect the fluorescence intensity of fluo-

rescein dye.

2692 APPLIED OPTICS / Vol. 14, No. 11 / November 1975

Artery _____

.. - I X -.

ta BS CUHR

vein

lnj8Cti~n 5 1 5 JV 20 25 3 5 Tm - Sec.injetn -sec 30

Fig. 4. Typical dilution curves recorded from an artery and a vein

in the superior temporal segment of the retina in a healthy subjectwith no ocular abnormalities.

segment of a normal subject's eye after an injectionof 30-mg fluorescein into the antecubital vein. Thedye appeared in the artery and the vein approximate-ly 10 sec and 11 sec, respectively, after the injection.It reached a maximum concentration at about 14.5sec in the artery and at about 17 sec in the vein. Theconcentration almost returned to the baseline withinapproximately 25 sec for the artery and 29 sec for thevein. At this time, however, the dye concentrationagain rose slightly, indicating that some of the dyecirculated a second time through the segment. Thisphenomenon is known as recirculation. The meancirculation time obtained from these curves using thestandard procedure of calculations is 3.7 sec. Similarrecordings obtained from the same subject within 30min gave MCT's of 3.6 sec, 3.7 sec, 3.3 sec, and 3.7sec.

VI. Discussion

The recordings shown in Fig. 4 represent the timecourse of fluorescence intensity Ia(t) and IV(t) from a

small portion of an artery and a vein of the retina.These curves are equivalent to arterial and venousdilution curves Ea(t) and cV(t), respectively, but only

if I(t) is proportional to c(t). (t) represents the

mean concentration of dye in the portion of the ves-sel under measurement. However, this equivalencecondition is not met in the case of blood, because theincident excitation light and the emitted fluorescentlight are strongly attenuated by hemoglobin. As aresult, the fluorescence emitted in the superficiallayers of the blood column contributes more effec-tively to the detected intensity than does the fluores-cence emitted in deeper layers. This leads to distort-

ed recording of the dilution curves ca(t) and a(t).This distortion is further accentuated by the nonuni-form distribution of the dye within the cross sectionof the vessel. This distribution varies in time, bothin the arteries and the veins. To evaluate this distor-tion, we have to know the cross-sectional distributionof the dye as a function of time. One can show thatthe following relation holds between I(t) and c(t):

I(t) = K0ro 2A(ro, CH, T, t) c(t). (1)

where T is a spatial function related to the geometryof the fiber aperature. It is valid only when fluores-cein moves in a laminar flow with a parabolic velocityprofile. In this equation, K is a constant of propor-tionality, Io the intensity of the excitation light at thefront surface of the vessel, ro the radius of the vessel,and CH the hemoglobin concentrations. The factorA(ro,CH,t) expresses the effect of attenuation of theincident and fluorescent light by hemoglobin. Itsvalue, always smaller than one, depends upon ro, CHand varies in time according to the cross-sectionaldistribution of the fluorescein. Tr) describes thetransmission of the aperture of the fiber. T(r) = 1for I rl < Ra and 0 elsewhere. Ra is the radius of theaperture. Explicit evaluation of Eq. (1) for dilutioncurves from main arteries and veins will be presentedin another paper.

We thank R. Thompson for modifying the Zeissfundus camera and building the detector assembly.We also thank F. Delori for helpful suggestions andF. Rogers for designing the electronic system. DavidF. Dobies assisted in preparing the manuscript.

This work was supported by Public Health ServiceResearch grant 1-RO1-EY-01242-01 from the Nation-al Eye Institute, National Institutes of Health, by theAdler Foundation, the Massachusetts Lions Eye Re-search Fund, Inc., and the Research to PreventBlindness William Friedkin Award for 1974.

References1. C. J. Bulpitt and C. T. Dollery, Cardiovasc. Res. 5, 406 (1971).2. D. H. Sliney and B. C. Freasier, Appl. Opt. 12,1 (1973).3. C. E. Riva and I. Ben-Sira, J. Opt. Soc Am. 63,1050 (1973).4. C. E. Riva, Appl. Opt. 11, 1845 (1972). -

5. A. C. Guyton, C. E. Jones, and T. G. Coleman, CirculatoryPhysiology: Cardiac Output and its Regulation (Saunders,Philadelphia, 1973), p. 43.

November 1975 / Vol. 14, No. 11 / APPLIED OPTICS 2693