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Ultrastructural examination (Fig. 6) confirmed a grant from Illinois Lions Club. Submitted for these findings, showing that all retinal layers were publication Jan. 3, 1975. Reprint requests: Dr. normal in the center of the biopsy specimens Gholam A. Peyman, University of Illinois with the exception of a partial vacuolization of and Ear Infirmary, 1855 W. Taylor St., Chicago, myelinated nerve fibers in the nerve fiber layer. 111. 60612. Discussion. Biopsy specimens taken with our Key words: chorioretinal biopsy, electron micros- technique show no significant structural and ultra- copy, sympathetic ophthalmia, retinitis pigmentosa, structural changes as a result of the procedure. uveitis. Despite peripheral ultrastructural changes, and the REFERENCES artifactual detachments noted in four specimens, all ten specimens were histologically normal in 1. Krill, A. E.: Retinitis pigmentosa: a review, central areas and adequate for both light and Sight Sav. Rev. 42: 21, 1972. 2. Carr, R. E., and Siegel, I. M.: Unilateral electron microscopic study. retinitis pigmentosa, Arch. Ophthalmol. 90: Surgical and postoperative complications ob- 21, 1973. served during the course of the experiment were 3. Witmer, R.: Etiology of uveitis, Ann. Oph- few in number. All observable remained thalmol. 4: 615, 1972. attached and appeared normal by ophthalmoscopic 4. Gouras, P., and Chader, C: Retinitis pig- and histologic observation. The corneal damage mentosa and retinal binding protein, INVEST. seen in two was the result of the heavy OPHTHALMOL. 13: 239, 1974. anterior chamber exudates. This may be avoided 5. Peyman, G. A., and Dodich, N. A.: Full- by treatment with anti-inflammatory drugs. Fur- thickness eye wall resection: an experimental approach for treatment of choroidal mel- thermore, it has been consistently observed in anoma. I. Dacron-graft, INVEST. OPHTHAL- this laboratory that the rabbit eye is more sensi- MOL. 11: 115, 1972. tive to surgical trauma than that of other ex- 6. Peyman, G. A., May, D. R., Ericson, E. S., perimental animals and that of man. The two et al.: Full-thickness eye wall resection: an cataracts, one only a small segmental one, might experimental approach for treatment of easily be avoided in a species with a smaller choroidal melanoma. II. Homo- and hetero- lens. The likely cause in this instance was post- graft, INVEST. OPHTHALMOL. 11: 668, 1972. operative hypotony and subsequent condensation 7. Peyman, G. A., Nelsen, P. T., Axelrod, A. J., of fibrin from secondary aqueous. et al.: Full-thickness eye wall resection: This procedure provides a method for studying evaluation of preoperative photocoagulation, unusual ophthalmologic diseases affecting the INVEST. OPHTHALMOL. 12: 962, 1973. 8. Peyman, G. A., Nelsen, P. T., Axelrod, A. J., and . The resulting tissue speci- et al.: Eye wall resection in primates: an mens will be available for histologic study with experimental approach to treatment of the electron and light microscopes, for biochemi- choroidal melanoma, Arch. Ophthalmol. 89: cal and immunologic analysis, and for tissue cul- 410, 1973. ture. Repeated biopsies could be used to follow 9. Peyman, G. A., Axelrod, A. J., and Graham, the progress of a disease such as animal models R. O.: Full-thickness eye wall resection: an of retinitis pigmentosa and other retinal dys- experimental approach for treatment of trophies. choroidal melanoma: evaluation of cry- With further refinement, this procedure could othermy, diathermy, and photocoagulation, Arch. Ophthalmol. 91: 219, 1974. bs used in the diagnosis and study of human 10. Peyman, G. A., and Apple, D. J.: Local chorioretinal disease. One indication for this pro- excision of choroidal melanoma: full-thick- cedure is the diagnosis of sympathetic ophthalmia, ness eye wall resection, Arch. Opthalniol. a condition that requires enucleation as a diag- 92: 216, 1974. nostic and therapeutic procedure. When this diagnosis is suspected in cases of blind eyes this procedure allows a tissue diagnosis that could Excitation and emission spectra of fluo- save eyes currently being enucleated. Diseases rescein dye in the human ocular . such as retinitis pigmentosa and uveitis, which have multiple etiologies and threaten permanent FRANCOIS C. DELORI AND ISAAC BEN-SIRA. visual loss, could be biopsied and studied with The excitation and emission spectra of fluo- emphasis on new therapeutic approaches that rescein dye were determined during could save remaining vision. Informed consent from different sites in the human ocular fundus. and philosophical judgment weigh heavily on All spectra were markedly shifted toward longer this aspect of the future use of this procedure. wavelengths relative to the spectra of in aqueous solution. This effect is most pronounced From the Department of , Uni- vsrsity of Illinois Eye and Ear Infirmary, Chicago, for the macular area; however, it decreases for the 111. Supported by Grant PHS EY 1107-02 from choroidal background and even more for the the National Institutes of Health, and in part by retinal vessels. The results are relevant to the

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selection of optical filter combinations for fluo- converted to exposure levels by using the calibra- rescein angiography. tion of each film with correction for the level of "pseudo " measured from control The spectral transmission bands of the exciter frames. The resulting fluorescence intensities for and barrier filters used routinely in fluorescein each site, denoted by F (exciter filter, barrier angiography have been selected 01; the basis of filter) were then transformed into relative excita- the excitation and emission spectra of fluorescein tion intensities A(X) by: in aqueous solution1 and a few in vitro measure- ments of the spectra of the blood-fluorescein mix- F(X, 22) 1(525) 2 4 A(X) = ture. " However, it is apparent that filters should F(525,22) ' I(X) be chosen with respect to the excitation and emission spectra of fluorescence from the fundus, where I(X) is the energy delivered to the sub- since both spectra are dependent not only upon ject's eye by an exciter filter centered at X. Rela- the characteristics of fluorescein in blood, but tive emission intensities E (X) were obtained from: also upon the spectral transmission properties of F(B4, X) T(525) S(525) the ocular tissues, which both the excitation light E(X) = and the emitted fluorescence must traverse. This F(B4,525) T(X) S(X) report presents in vivo measurements of the ex- where T(X) is the integrated transmission of the citation and emission characteristics of the fluo- barrier filter centered at X and S(X) the spectral rescence from retinal and , the sensitivity of the film at X. choroidal background, and the macular area, made The excitation and emission spectra of fluo- during dye transit. rescein in aqueous solution (concentration 3 x 10~G Methods. Four Caucasians with normal vision mg. per milliliter, pH 7) were measured under and no ocular abnormalities volunteered for several frontal illumination using standard spectrofluo- angiography experiments. The subjects were a rimetric techniques. Convolution of these spectra 38-year-old male (No. 1), an 18-year-old female with the transmission characteristics of the narrow (No. 2), a 24-year-old female (No. 3), and a band filters yielded relative values of A(X) and 35-year-old female (No. 4). Fundus pigmentation E(X) for fluorescein in aqueous solution. ranged between light (No. 1) to very dark (No. Results. The results of the measurements of 4). The fluorescein dye (500 mg. in 5 ml.) was A(X) and E(X) for sites in the fundi of the injected in the antecubital . different subjects are given in Table I, along with The excitation spectra were measured by com- the relative values for fluorescein in aqueous solu- paring the intensity of fluorescence emission tion. The values of A(X) and E(X) for the veins, through a Kodak Wratten 22 barrier filter (trans- arteries, and choroidal background of the individ- mission 550 to 700 nm.) for four narrow spectral ual subjects were obtained by averaging at least bands of excitation, centered at 450, 475, 500, five measurements of different vessels and the and 525 nm. The emission spectra were measured surrounding background. Although large variations by inducing fluorescence with a B4 filter (trans- occurred in absolute intensity at these sites, the mission 450 to 500 nm.) and comparing the inten- relative intensities A(X) and E(X) were always sities of emission in five narrow spectral bands, within 15 per cent of their average value for centered at 525, 550, 570, 590, and 610 nm. All retinal vessels and within 25 per cent for the back- narrow band filters had a half-width of about 25 ground. Graphic representation of the results from nm. A Zeiss fundus camera, with an amplified one subject are shown in Fig. 1. flash, was used to record the angiograms with Excitation spectra. Compared to the excitation either alternating exciter filters or alternating spectrum of fluorescein in aqueous solution, the barrier filters at a rate of about one frame every excitation spectra of the fluorescence from the 2.0 seconds. The Kodak Tri-X film was developed fundus are characterized by a marked reduction in Kodak D19 (dilution 1:1) for 10 minutes at of the relative excitation intensities A (X) at the 20° C. short wavelength end of the spectra. The maxi- The exposed negatives were analyzed with a mum excitation wavelength is shifted from about Joyce Loebl Microdensitometer. The density of 485 nm. for aqueous solution to around 500 nm. large retinal veins and arteries near the disc, and for the various sites in the fundus. The resulting the density of the surrounding choroidal back- shift of the spectrum toward longer wavelengths ground and fovea were measured in successive is most pronounced for the fovea; this shift, how- frames corresponding to known filter combinations. ever, decreases for the background and even more All measurements were made after complete filling for the retinal arteries and veins. These trends of the retinal veins, but not later than one minute were found in all subjects (Table I). after injection. The slow decay of the fluorescein The continuous change in the shape of the dye concentration during that time interval was excitation spectrum in the fundus is illustrated taken into account in the analysis. Densities were by the variation of the ratio A(475) (see Fig. 2)

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Table I. Relative excitation and emission intensities for the fluorescence from the retinal veins (V), the retinal arteries (A), the choroidal background (B) and the fovea (F). The values A(X) represent the intensities of fluorescence emission above 550 nm. for equal energy of excitation at X. The values E(X) are the intensities of emission at X for an excitation in the spectral band 450 to 500 nm. The equivalent data for fluorescein in aqueous solution are given for comparison. All values of A(X) and E(X) are normalized at 525 nm.

Subjects Aqueous solution Sites No. I No. 2 No. 3 No. 4 V 0.88 1.05 1.52 A(450) A 0.75 0.80 1.22 5.25 B 0.50 0.65 0.85 F 0.21 0.26 0.41 V 2.41 2.87 3.20 A(475) A 2.12 2.45 2.75 11.50 B 1.80 2.05 2.53 F 0.82 1.05 1.47 V 4.96 5.40 6.05 A(500) A 4.69 5.05 5.52 8.50 B 4.00 4.35 4.81 F 2.63 2.94 3.23 A(525) and 1.00 1.00 for all sites E(525) V 0.51 0.55 0.52 E(55O) A 0.50 0.51 0.53 0.34 B 0.58 0.60 0.59 F 0.70 0.72 0.66 V 0.25 0.31 0.29 E(57O) A 0.23 0.29 0.28 0.14 B 0.30 0.36 0.34 F 0.36 0.44 0.42 V 0.16 0.18 0.15 E(590) A 0.16 0.19 0.16 0.07 B 0.21 0.20 0.18 F 0.24 0.24 0.22 V 0.17 0.16 0.14 E(610) A 0.19 0.18 0.16 0.14 B 0.20 0.19 0.16 F 0.23 0.23 0.19

along a line through the fovea in the superonasal other sites. Similar changes were found in all inferotemporal direction. The ratio is lowest in the subjects (Table I), with smaller individual varia- fovea, but increases—first rapidly up to about 5° tion than for A (X ). from the fovea, and then slowly—as one moves Discussion. The predominant characteristic of further away from the macular region. Whenever the fluorescence spectra from the fundus, com- the trace crosses a retinal vessel, this ratio in- pared to the spectra of fluorescein in aqueous solu- creases steeply. tion, is a shift of both the excitation and emission Emission spectra. Compared with the spectrum spectra toward longer wavelengths. This shift is of fluorescein in aqueous solution, the emission most marked for the fovea and decreases for the spectra of fluorescence from the fundus (Fig. 1) choroidal background and even more for the are also characterized by a shift toward longer retinal vessels. wavelengths. Because the results were normalized The spectra at all sites in the fundus are af- at 525 nm., this shift is revealed by an increase fected by the rapid increase in the transmission in the relative intensities E(X) at longer wave- of the ocular media between 400 and 540 nm.7 lengths. The shift of the entire spectrum is again and, except for the fovea, by the loss of light by most pronounced in the fovea, and decreases for scattering at short wavelengths in the nerve fiber the background and even more for the retinal layer. vessels. All spectra are characterized by a flatten- The spectra of fluorescein in blood-"1 measured ing of their shape above 570 nm. and this effect in vitro exhibit some shift toward longer wave- is more marked for retinal arteries than for the lengths compared to the spectra in aqueous solu-

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EU)

EMISSION SPECTRA

10 1.0

0.1

0.1 525 WAVELENGTH (nm.) Fig. 1. Excitation and emission spectra from retinal veins (V), retinal arteries (A), choroidal background (B), and fovea (F) for subject No. 3, compared to the spectra of fluorescein in aqueous solution (AS). Spectral resolution is about 25 nm.

tion, but these data are not consistent enough to the shift of the spectra, more so for arteries to permit comparison with our results. The exci- than for the veins, which have thinner walls. tation spectmm of the fluorescein molecules bound With increasing wavelength, the transmission of to albumin was reported" to be reduced in mag- the retina and pigment epithelium increases nitude and shifted toward longer wavelengths, throughout the visible spectrum,7 because absorp- compared to the spectrum of free fluorescein mole- tion by melanins and light scattering decrease. cules. The absorption of the excitation and emitted This undoubtedly contributes to the shift toward light by hemoglobin5 (inner filtering) reduces longer wavelengths in the excitation and emission the efficiency of fluorescein in blood and also spectra of the choroidal and foveal fluorescence. affects the shape of the spectra. The higher ex- Since the measurements were performed during tinction coefficient of oxygenated hemoglobin as the venous phase of the dye transit, part of the compared to reduced hemoglobin between 450 to choroidal fluorescence emanates from extravasated 500 nm. and the near equality of these coefficients fluorescein dye, which stains Bruch's membrane. around 525 nm. may contribute to the larger shift During the early stage of the dye transit, a larger toward longer wavelengths in the excitation spec- spectral shift in the choroidal fluorescence toward trum of arteries as compared to veins. Also, the long wavelengths is expected, since the dye is then flattening of the emission spectra above 570 nm. located in deeper layers. The excitation and may be caused by the rapid decrease in absorption emitted light is therefore more affected by scatter- of emitted light by hemoglobin between 580 and ing and absolution in Bruch's membrane and the 620 nm. This flattening is more pronounced for choroid. the arteries than the veins, because the decrease The macular dark spot observed in routine in the extinction coefficient for oxygenated hemo- fluorescein angiography is caused by the increased globin is more abrupt than for reduced hemoglobin. density of visual pigments and melanin granules Scattering in the vessel walls may also contribute and by the presence of xanthophyll in the inner

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A(475) X9 = 518 ± 5 nm. for retinal veins and for Xs = 528 ± 7 nm. for the fovea. Compared to the best available filter combination (SE4 excitation filter 450 to 495 nm., SB5 barrier filter 495 to 620 nm.), a filter combination with a separation wavelength at 525 nm. (excitation filter 450 to 525 nm., barrier filter 525 to 620 nm.) would provide approximately a twofold increase in the fluorescence intensity from the retinal veins and a threefold increase from the fovea. Preliminary trials of such new filters on normal subjects have confirmed these predictions.10 Because the gain in intensity is higher for the choroidal fluorescence than for the retinal vessels, the new filters provide a much improved visualization of the choroidal vasculature. The authors are indebted to Dr. Charles E. Riva for his encouragement throughout this project. Expert technical assistance was provided by Mr. John Trovato and Ms. Julianne Schneider. Editorial assistance was provided by David F. Infero- Supero- Dobies, M.A. temporal nasal From the Eye Research Institute of Retina Foundation, Boston. This work was supported in 10 5 0 5 10 15 part by Public Health Service Research Grant DISTANCE from FOVEA (degrees) EY-00227 from the National Eye Institute, Na- tional Institutes of Health, and by the Research Fig. 2. Variation along a line passing through the to Prevent Blindness, William Freidkin Award fovea of the ratio between the fluorescence inten- for 1974. Submitted for publication Dec. 3, 1974. sity excited at 475 nm. and that excited with Reprint requests: Editorial Services Unit, Eye equal energy at 525 nm. (subject No. 1). Research Institute of Retina Foundation, 20 Staniford St., Boston, Mass. 02114. retinal layers. Xanthophyll extends to about 5° Key words: fluorescein angiography, fluorescein from the fovea, substantially absorbs light between excitation spectra, fluorescein emission spectra, 440 and 490 nm., and becomes practically trans- filters for angiography, optical properties of ocular parent above 520 nm.9 Hence, it contributes to the tissue. shift toward the long wavelengths of the excita- REFERENCES tion spectrum in the foveal area and to the marked absorption of the excitation light at 475 1. Wessing, A.: Fluorescein Angiography of the Retina. Saint Louis, The C. V. Mosby Com- nm. (Fig. 2). pany, 1969, p. 14. Practical conclusions. The overall efficiency of 2. Allen, L., and Frazier, O.: Evidence favoring fluorescence from the fundus depends upon the wide band filters for fluorescein angiography. choice of the separation wavelengths Xs between Proceedings of the International Symposium the spectral ranges of transmission of the excita- on Fluorescencs Angiography Albi, 1969. tion filter and the barrier filter. Consider an ideal Basel, 1971, S. Karger. exciter filter (100 per cent transmission between 3. Haining, W. M., and Lancaster, R. C: Ad- vanced techniques for fluorescein angiography, 450 nm. and Xs) used in conjunction with a barrier filter (100 per cent transmission between Arch. Ophthalmol. 79: 10, 1968. 4. Hodge, J. V., and Clement, R. S.: Improved Xs and 620 nm.). The overall efficiency is then method for fluorescence angiography of the proportional to: retina, Am. J. Ophthalmol. 61: 1400, 1966. X 620 nm. 5. Horecker, B. L.: The absorption spectra of B hemoglobin and its derivatives in the visible J I(X)A(X)dX • J E(X)S(X)dX and near infra-red regions, J. Biol. Chem. 450 nm. Xs 148: 173, 1943. where I(X) is the energy of the light source at X 6. Laurence, D. J. R.: A study of the absorption (xenon arc) and S(X) the spectral sensitivity of of dyes on bovine serum albumin by the the film (Kodak Tri-X, standard development). method of polarization of fluorescence, Bio- The above expression was evaluated by numerical chem. J. 51: 168, 1952. 7. Geeraets, W. J.: The relative absorption of integration of the experimental spectra for dif- thermal energy in retina and choroid, INVEST. ferent values of X8. The results indicate that the OPHTHALMOL. 1: 340, 1972. highest efficiency of fluorescence is obtained for 8. Meyer-Arendt, J., and Bayer, M.: Absorptions-

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spektrum von Melaninpigment, Experientia molecule and possibly a 208Pb-Tris complex, may 10: 371, 1954. penetrate cells, and may neutralize acidic ions in 9. Wald, C: Human vision and the spectrum, the intracellular fluid. The formation constant of Science 101: 653, 1945. Pb+2 with Tris was expected to be small. It was 10. Delori, F. C, Ben-Sira, I., and Trempe, C: hypothesized9 that the 203Pb might become Improved filters for fluorescein angiography. physiologically associated in tumor as a metal- In preparation. melanin-protein complex of the type predicted by Bowness and Morton.10 Further details are found elsewhere.11 Localization of radioactive lead in ocular Methods. Skin melanomas were maintained with and skin melanoma. S. PACKER, R. M. the Greene melanoma in male Syrian golden LAMBRECHT, J. C. MERRILL, H. L. ATKINS, hamsters by subcutaneous transfer. All tumors AND A. P. WOLF. were pigmented, i.e., melanotic. Transplantation took place between the second and third week. The ability of radioactive lead to localize For ocular melanoma, a cell suspension was made melanomas was studied. The Greene melanoma from 1.0 Gm. of the Greene melanoma which in the Syrian Golden hamster served as a model was cut into small pieces, mixed with 5.0 ml. of for both skin and ocular melanoma. The affinity tissue culture media and forced through a micro- of heavy metals for neoplasms has been studied sieve (27 micron pores). There were approxi- but previous reports have been inconsistent as to mately 50 cells per 5.0 /A. A straight pin was used tumor specificity. For this investigation the radio- to puncture both and sclera. Swirling active lead (203Pb,) was studied as the chemical the pin against a firm allowed a track to be complex s03Pb-Tris. Significant tumor-.nontumor made through the sclera while avoiding puncturing ratios were found in ocular melanoma and the of the globe and entry into the vitreous. A 30- concentration in the lens was minimal. The ratio gauge needle was inserted into the track and of per cent uptake per gram of tumor: per cent passed tangentially beneath the sclera for a short uptake per gram in control tissue with skin distance. Five microliters were injected and while melanoma was 9.4 at 24 hours and for the eye the needle was being withdrawn Eastman 910 melanoma the ratio was 26.3 at 24 hours. The glue was applied to the wound. Antibiotic oint- affinity of Z0SPb-Tris for melanomas appears to be ment was applied at the conclusion of this pro- as promising as other compounds presently being cedure. evaluated for ocular > namely, labeled quinoline analogs. Therefore, further preclinical Carrier-free 203Pb was produced on the BNL evaluation is warranted. 60 inch cyclotron using the 203Tl (d,2n) 203Pb nuclear with the deuterons degraded from Although inorganic metals such as Zn,1- 2 Cu,3 22.7 —> Q MeV. The experimental method includ- Hg,4 and Pb5> 6 have been evaluated for tumor ing the chemical separation of the 203Pb from the specificity, radioactive lead does not appear to target of naturally occurring thallium metal has have been looked at specifically for the purpose been described.8 Carrier-free 203Pb-Tris was of melanoma localization. Lead-203 (203Pb) is a prepared by addition of an aqueous solution con- radioactive nuclide of lead that has excellent taining 2-amino-2-hydroxymethyl 1,3-propanediol physical characteristics appropriate for tumor to the carrier-free 203Pb. The ligand is also known scanning. It has a short half-life, 52.1 hours, and as Tris, and is commonly used in Tris and THAM- decays by electron capture with the emission of E™ (Abbott Laboratories, North Chicago, 111.) a high abundance (95 per cent) of 279 keV biological buffer. The pH was adjusted to 6.8 to 7.2. photons, and the absence of any beta radiation. Specimens were placed in preweighed vials and The tissue penetration of its photon radiation is radioactivity was measured in vitro using an auto- sufficient for localization of deep-seated tumors, matic gamma-counter (1185) series (Nuclear- specifically posteriorly located choroidal melanoma. Chicago, Des Plaines, 111.). All samples were This would obviate the necessity for invasive reweighed again immediately after dissection on techniques of localization. A low uptake by the an analytical balance. The data were corrected for retina implies that there would be little back- radioactive decay and the results presented as ground interference, while a low uptake by the the per cent uptake per gram of tissue. lens would indicate the safety of the radionuclide Control eyes were those that had no tumor. with regard to the induction of a radiation Where the weight of the individual specimen was cataract. Preliminary preclinical studies using the small, as with the different parts of the eye, a Greene melanoma in hamsters have shown sig- total of four eyes were used. Thus the nificant tumor specificity of 203Pb-Tris. uptake is the average of four control . The rational for selecting Tris9 as the carrier Results. Table I gives the tissue distribution of was that at pH 7.4, 30 per cent of compound is 203Pb-Tris in eye melanoma and the various parts not ionized and therefore is capable of reaching of the eye. The ratios of eye melanoma to normal equilibrium in total body water. The nonionized eye is 26.3:1 at 24 hours although 6 hours is the

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