Volume 14 Reports 487 Number 6 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 Eye 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 retinas 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 eyes 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., retina and choroid. 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 fundus. 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 angiography 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 fluorescein in aqueous solution. This effect is most pronounced From the Department of Ophthalmology, 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 Downloaded from iovs.arvojournals.org on 09/26/2021 488 Reports Investigative Ophthalmology June 1975 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 fluorescence" 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 veins and arteries, 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 vein. 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.