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Topographic distribution of refractive indices in bovine lenses
Exp. Eye Res. (1974) 18, 351-356
Topographic Distribution of Refractive Indices in Bovine Lenses FREDERICK
A. BETTELHEIM
AND TAILER
J. Y. WANG
Chemistry Department, Adelphi University, Garden City, N. Y. 11530, U.S.A. (Received27 July 1973, and in revisedform 10 December1973, Boston) The surface of the bovine lens was mapped for refractive indices by using the minimum of light scattered as a probe in different non-interacting media with different refractive indices. The highest refractive index is at the center and the refractive indices decrease symmetrically moving toward the corner of the lens along a horizontal axis. The same is true along the vertical axis. It seems that in order to provide a uniform refractive power of the whole lens the change in the curvature is compensated by a change in the refractive index.
1. Introduction In our previous studies on the light scattering of bovine lenses (Vinciguerra and Bettelheim, 1971, and Bettelheim, Vinciguerra and Kaplan, 1973) thin slices were used in order to avoid the difficulty of the interpretation of multiple scattering. This led to the understanding of the alignment and organization of the fiber cells in static and dynamic conditions but did not allow the integration of the different scattering patterns of the slicesinto a total light-scattering pattern of the whole lens. In our continued effort to understand the nature of scattering of the whole lens, we thought to simplify our study by separating the surface scattering from the bulk scattering by eliminating the surface scattering of the isolated lens. The surface scattering per seis an important problem in vivo since the aqueoushumor on the one hand and the vitreous on the other differ greatly from the lens as far as the refractive index is concerned (Kuck Jr., 1970). Our assumption was that when the isolated bovine lens is submerged in a non-interacting liquid which matches the refractive index of the surface layer of the lens one obtains a minimum scattering. Our assumption wa,s proven by experiments. As a result of these investigations, however, we found that the minimum of light scattered by the lens can be usedas a criterion to find the refractive indices at different loci.
2. Materials and Methods Adult bovine eyes (2 years old) were obtained from a slaughterhouseand used 1 day or less, post mortem. The lens capsule was removed together with the adhering epithelial cells. The lens was then placed in a glass cuvette carefully noticing the positions of the different topographic directions, i.e. anterior, posterior, upper and lower. The non-interacting liquid immersion media were made of mixtures of hexamethyldisiloxane (ns5 = 1.3722) and butylphthalate (n 25 = 1.4882). Hexamethyldisiloxane was obtained from PCR, Gainsville, Fl. ; it was used directly. Butylphthalate was obtained from Consolidated Vacuum Corp., Rochester, N.Y. ; it was purified by vacuum distillation. In selecting non-interacting immersion fluids, the following criteria were used. (a) The medium should be apolar so that water, ions and polar organic molecules should be insoluble in it, (b) The medium should not be able to penetrate into the fiber cells or their cell walls. Hexamethyldisiloxane and butylphthalate were found to fulfil condition under 351
Xr!
ii.
.I.
I~E7”l’l~:I,IIC:I\I
ANI)
‘I’.
.I.
\-.
\\‘AK((:
(;I) (Gilles, 1968). Moreover t’he\- werr con~pletelv miscible within the refra&\-c iurlex ravage recluired. The immersion medium (lid not penetrate the fiber cells am1 the membranes. This was clemonst,rat,ed by microscopic ohservatiou of t,he cortex immersecl in the liquids over a 24-hr period as well as monit,oring the light, seat.tering p&tern of :L lens immersed during a 24.hr period, starting with the first exposure, 2 3 set after t.lie ifnruersion. No change was detected with either technique during the 24-1~ period. The cuvette containing the lens was filled with hesamethyldisilosane t,o sufficient height to cover the lens. The cuvette was placed on an optical t)enc:h. A He+Ke laser (Spectra Physics 132) was used which provided a vertically polarized light) of 1 mm in tliamet,er. The cuvette was arranged so t,hat the laser beam was normal to the surfacse of the lens at’ any selected topographic position of the lens. Behind t,he cuvette a Polaroid was placed which was eit,her aligned parallel to the direction of polarization (I’,. mode) or perpendicular to it (H, mode). The scattering patterns mere recorded photographically at a set sample to film distance (40 cm) by using Kodak Plus X Pan 10.2 ‘< 12.7 cm film in a cassette on the optical bench. Once a set of scattering patterns (H,! and /1J \vere recorded in the first immersion liquid ; butylpht,halate was added and mixed t,horoughly iu order to obtain the immersion liquid with a higher refractive index. Care ~-as t,aken to allow true equilihration with the new surrounding and to assure that the lens did not, move from its previ:ms position. A new set of scat,tering patterns (H, and J’?.) were t,aken aud the process was repeated through the whole refractive index range covered by the two immersion licluid (&Jj = 1.3722-+ = 1.4882). Once t’his was completed the cuvette was aligned so that a new area of the lens was exposed normal t#o t,he laser beam and the whole process covering the refractive index range was repeated. In this manner the whole lens was mapped. Blank laser scattering patterns caused by the cuvette and the immersion liquids alone were also recorded. All the films were developed and fixed under identical contiitions. Previous calibration experiments employing neutral filters proved that the intensit,! readings by densitometer of the expose11 films were proportional t.o the intensities of the light beam. Direct refractive index measurements were also taken on different sections of the isolated decapsulated bovine lens hy simply separating t’he lens into the cortex and nucleus and sectioning small parts of each and putting the sections in au Abbi refractometer using white light. _ Similar measurements were attempted on the lens without removing the lens capsule. It was found however, that the hesamethyldisilosane, the non-interacting liquid with the lowest refractive index, was still too high to match the refractive index of the capsule intact on the lens. In order t’o ascertain the refractive index of the capsule the isolated capsule was spread as a film in the Sbbi: refractometer. Furthermore, an approximate mapping of the refractive index of the isolated lens capsule was attempt’ed by sect’ioning the capsule into small 2 ;< 2 mm squares and quickly suspending them in solutions with adjusted sucrose concent,rations until they were not. visible. The refractive index of the media then was taken as that of the capsule segment. During the quick refrac,tive index matching process no appreciable swelling or shrinking of the lens capsule occurred. This was proven by the fact that the average refractive index of the capsule measured in the refractometer a.nd that of the lens capsule segments suspended in sucrose solutions were substantiallv the same.
3. Results Figure 1 shows the change in t,he light, scattering pattern when an optic glass lens was immersed in liquids with different refractive indices. The minimum scattering was obtained when the immersion liquid matched the refractive index of the glass lens (E = 1.4422). Thus, the minimum light scattering technique can be used to find
FIQ. 1. Laser scattering patterns of a glass lens (n = 1.4422) in different immersion media with normal to the surface of the lens and the scattering patterns are in the b’, mode.) The refractive Ic) 14122: (d) 14850.
different refractive indices. (The laser beam was indices of the media were (a) 1.3544; (b) 1.3940;
354
F. A.
BETTELHEIM
AND
T.
J.
Y.
WANO
the refractive index at the surface of the transparent lens of the eye. This technique would not be accurate with a lens having cataract, since the minimum of the multiple light scattering caused by the cataract would be a function of the refractive index of the immersion liquid. The average refractive index of the lens capsule as measured in the Abbe refractometer was 1.335. The refractive index of the lens capsule segments as measured by matching the refractive index of sucrose solutions varied between 1*339-1.340. No significant trend was found in the distribution of refractive indices along the lens capsule. Hence the lens capsule with the adhering epithelial cells may be taken as a film with uniform refractive index. The refractive indices of the surface of the decapsulated bovine lens at different topographic positions were obtained by the minimum light scattering technique. In Fig. 2 the distribution of these refractive indices along the anterior part of the lens equator is given. Similar distribution along the posterior surface is expected except in a narrower range.
FIG. 2. Topographic distribution of refractive indices in bovine lenses (top view). The staggered line is the projected pathway of the laser beam through the lens. The bracketed numbers are the refractive indices obtained by direct measurements of the refractive index of different sections. The non-bracketed numbers are the refractive indices obtained by the minimal light scattering technique.
The reason for this statement is the fact that the nucleus has a higher refractive index than the cortex, (Philipson, 1969) thus the projected light path deviates from the straight line forming a hyperbole (Fig. 2 staggered line). The matching of the refractive index of the immersion liquid with that of the lens surface must be the average of the anterior and posterior surface refractive indices. The results indicate that the highest refractive index is in the middle and the lowest refractive indices are at the corners and that there is a smooth transition between the two extremes. Direct refractive index readings on selected sections of the cortex of bovine eyes show sufficiently good agreement with the data obtained from light scattering. As was expected, the nucleus has higher refractive indices than the cortex, and again shows a tendency of having higher refractive index at the center and lower at the corner of the lens nucleus. The same can be said of the refractive index change along the vertical axis of the lens (Fig. 3).
REFRACTIVE
FIG. 3. Topographic
distributim
INDICES
of rrfractive
IN
BOVINE
indices
ill hake
LENSES
lenses (frontal
view).
4. Discussion The primary role of the lens in the visual process is focusing the image on the retina. This is achieved by refraction. The refractive power is due (a) to the difference between the refractive indices at the interface and (b) to the curvature of the lens. The lens is embedded in front in the aqueous humor and posterior in the vitreous. Both have refractive indices of 1.336 (Kuck Jr., 1970). The average refractive index of the lens is given as 1.420 (Lerman, 1964). Our measurements indicate that the lens capsule almost matches the refractive index of its naturally-embedding media the vitreous and the aqueous. The average value of 1.335 found in the Abbe refractometer is probably somewhat low because of the incomplete coverage of the whole prism area. The values of 1.339-1.340 found by the immersion technique were quite reproducible and it showed that the lens capsule with the adhering epithelial cells is essentially uniform as far as refractive index is concerned. The results in Fig. 2 show that using as a probe, the minimum light scattering obtained in different non-interacting immersion media with different refractive indices, one is able to map the local refractive indices along the lens surface. The direct measurements of refractive index on cortical and nuclear sections indicate the same trend as that obtained by light scattering. These results are especially heartening considering the fact that in the direct measurements of the cortical slice not only the surface fiber cells contributed to the refractive index but a large number of fiber cells under the surface were also averaged in the measurements. The higher refractive index in the nucleus compared to that of the cortex has been known for some time (Philipson, 1969). The mapping of protein concentrations of the lens along the optic axis by microradiography showed a gradual increase in protein content from cortex to the center of the nucleus in all but the youngest (few days old) rat lenses. What this study has established is that in decapsulated bovine lenses there is a gradual and symmetrical change in refractive indices going from the center toward the corner along the horizontal axis as well as along the vertical axis. This means that the highest surface refractive index is at the position where there is the least curvature in the lens and vice versa. Since the refractive power of the lens originates both
356
I-‘. A. 1~ETTELHEIY
ANI)
T.
J.
Y.
%‘ANC,
from the curvature as well as from the refractive index difference at, the interface, it. seems that the adaptive process in the evolution tried to obtain a lens with more or less uniform refractive poffer along the whole organ; thus the increase in curvature was compensated for by t,he decrease in refractive index. Since the refract,ive index is due largely to the concentration of the crystallins in the fiber cells, our finding JVOUM imply that the highest concentration per unit volume of these proteins are in the central area of the lens within each fiber all along the surface. At the same time. however, we found previously that the width of the fiber cells is narrower along the anterior-posterior surface than at the corner (Bettelheim and Vinciguerra, 1971), SO it is quite possible that the concentration of crystallins along a unit length of a fiber cell is uniform throughout the lens and only the width of the fiber cell. i.e. its packing is changing. ACKNOWLEDGMENTS
This research was supported by a PHS grant 1J.S. Public Health Services.
EY-00501-(J4
of the National Eye Institute,
REFERENCES Bettelheim, F. A. and Vinciguerra, 11. J. (19il). Ann. N.I’. dead. 81%. 177,427. Bettelheim, F. A., Vinciguerra, M. J. and Kaplan, D. (1973). E.rp. Eye Res. 15, 149. Gilles, P. (1968). Double Liaison 152,45i. Ku& Jr., J. F. R. (1970). In Biochemistry qf the Eye (Ed. Graymore, C. K.). P. 190. Academic Press, New York. Lerman, S. (1964). Cataract. Thomas. Springfield, Ill. Philipson, B. (1969). Incest. Ophthdmol. 8, 3. Vinciguena. M. J. and Bet,telheim, F. A. (1971). Exp. Eye Res., 11, 214.
Topographic Distribution of Refractive Indices in Bovine Lenses FREDERICK
A. BETTELHEIM
AND TAILER
J. Y. WANG
Chemistry Department, Adelphi University, Garden City, N. Y. 11530, U.S.A. (Received27 July 1973, and in revisedform 10 December1973, Boston) The surface of the bovine lens was mapped for refractive indices by using the minimum of light scattered as a probe in different non-interacting media with different refractive indices. The highest refractive index is at the center and the refractive indices decrease symmetrically moving toward the corner of the lens along a horizontal axis. The same is true along the vertical axis. It seems that in order to provide a uniform refractive power of the whole lens the change in the curvature is compensated by a change in the refractive index.
1. Introduction In our previous studies on the light scattering of bovine lenses (Vinciguerra and Bettelheim, 1971, and Bettelheim, Vinciguerra and Kaplan, 1973) thin slices were used in order to avoid the difficulty of the interpretation of multiple scattering. This led to the understanding of the alignment and organization of the fiber cells in static and dynamic conditions but did not allow the integration of the different scattering patterns of the slicesinto a total light-scattering pattern of the whole lens. In our continued effort to understand the nature of scattering of the whole lens, we thought to simplify our study by separating the surface scattering from the bulk scattering by eliminating the surface scattering of the isolated lens. The surface scattering per seis an important problem in vivo since the aqueoushumor on the one hand and the vitreous on the other differ greatly from the lens as far as the refractive index is concerned (Kuck Jr., 1970). Our assumption was that when the isolated bovine lens is submerged in a non-interacting liquid which matches the refractive index of the surface layer of the lens one obtains a minimum scattering. Our assumption wa,s proven by experiments. As a result of these investigations, however, we found that the minimum of light scattered by the lens can be usedas a criterion to find the refractive indices at different loci.
2. Materials and Methods Adult bovine eyes (2 years old) were obtained from a slaughterhouseand used 1 day or less, post mortem. The lens capsule was removed together with the adhering epithelial cells. The lens was then placed in a glass cuvette carefully noticing the positions of the different topographic directions, i.e. anterior, posterior, upper and lower. The non-interacting liquid immersion media were made of mixtures of hexamethyldisiloxane (ns5 = 1.3722) and butylphthalate (n 25 = 1.4882). Hexamethyldisiloxane was obtained from PCR, Gainsville, Fl. ; it was used directly. Butylphthalate was obtained from Consolidated Vacuum Corp., Rochester, N.Y. ; it was purified by vacuum distillation. In selecting non-interacting immersion fluids, the following criteria were used. (a) The medium should be apolar so that water, ions and polar organic molecules should be insoluble in it, (b) The medium should not be able to penetrate into the fiber cells or their cell walls. Hexamethyldisiloxane and butylphthalate were found to fulfil condition under 351
Xr!
ii.
.I.
I~E7”l’l~:I,IIC:I\I
ANI)
‘I’.
.I.
\-.
\\‘AK((:
(;I) (Gilles, 1968). Moreover t’he\- werr con~pletelv miscible within the refra&\-c iurlex ravage recluired. The immersion medium (lid not penetrate the fiber cells am1 the membranes. This was clemonst,rat,ed by microscopic ohservatiou of t,he cortex immersecl in the liquids over a 24-hr period as well as monit,oring the light, seat.tering p&tern of :L lens immersed during a 24.hr period, starting with the first exposure, 2 3 set after t.lie ifnruersion. No change was detected with either technique during the 24-1~ period. The cuvette containing the lens was filled with hesamethyldisilosane t,o sufficient height to cover the lens. The cuvette was placed on an optical t)enc:h. A He+Ke laser (Spectra Physics 132) was used which provided a vertically polarized light) of 1 mm in tliamet,er. The cuvette was arranged so t,hat the laser beam was normal to the surfacse of the lens at’ any selected topographic position of the lens. Behind t,he cuvette a Polaroid was placed which was eit,her aligned parallel to the direction of polarization (I’,. mode) or perpendicular to it (H, mode). The scattering patterns mere recorded photographically at a set sample to film distance (40 cm) by using Kodak Plus X Pan 10.2 ‘< 12.7 cm film in a cassette on the optical bench. Once a set of scattering patterns (H,! and /1J \vere recorded in the first immersion liquid ; butylpht,halate was added and mixed t,horoughly iu order to obtain the immersion liquid with a higher refractive index. Care ~-as t,aken to allow true equilihration with the new surrounding and to assure that the lens did not, move from its previ:ms position. A new set of scat,tering patterns (H, and J’?.) were t,aken aud the process was repeated through the whole refractive index range covered by the two immersion licluid (&Jj = 1.3722-+ = 1.4882). Once t’his was completed the cuvette was aligned so that a new area of the lens was exposed normal t#o t,he laser beam and the whole process covering the refractive index range was repeated. In this manner the whole lens was mapped. Blank laser scattering patterns caused by the cuvette and the immersion liquids alone were also recorded. All the films were developed and fixed under identical contiitions. Previous calibration experiments employing neutral filters proved that the intensit,! readings by densitometer of the expose11 films were proportional t.o the intensities of the light beam. Direct refractive index measurements were also taken on different sections of the isolated decapsulated bovine lens hy simply separating t’he lens into the cortex and nucleus and sectioning small parts of each and putting the sections in au Abbi refractometer using white light. _ Similar measurements were attempted on the lens without removing the lens capsule. It was found however, that the hesamethyldisilosane, the non-interacting liquid with the lowest refractive index, was still too high to match the refractive index of the capsule intact on the lens. In order t’o ascertain the refractive index of the capsule the isolated capsule was spread as a film in the Sbbi: refractometer. Furthermore, an approximate mapping of the refractive index of the isolated lens capsule was attempt’ed by sect’ioning the capsule into small 2 ;< 2 mm squares and quickly suspending them in solutions with adjusted sucrose concent,rations until they were not. visible. The refractive index of the media then was taken as that of the capsule segment. During the quick refrac,tive index matching process no appreciable swelling or shrinking of the lens capsule occurred. This was proven by the fact that the average refractive index of the capsule measured in the refractometer a.nd that of the lens capsule segments suspended in sucrose solutions were substantiallv the same.
3. Results Figure 1 shows the change in t,he light, scattering pattern when an optic glass lens was immersed in liquids with different refractive indices. The minimum scattering was obtained when the immersion liquid matched the refractive index of the glass lens (E = 1.4422). Thus, the minimum light scattering technique can be used to find
FIQ. 1. Laser scattering patterns of a glass lens (n = 1.4422) in different immersion media with normal to the surface of the lens and the scattering patterns are in the b’, mode.) The refractive Ic) 14122: (d) 14850.
different refractive indices. (The laser beam was indices of the media were (a) 1.3544; (b) 1.3940;
354
F. A.
BETTELHEIM
AND
T.
J.
Y.
WANO
the refractive index at the surface of the transparent lens of the eye. This technique would not be accurate with a lens having cataract, since the minimum of the multiple light scattering caused by the cataract would be a function of the refractive index of the immersion liquid. The average refractive index of the lens capsule as measured in the Abbe refractometer was 1.335. The refractive index of the lens capsule segments as measured by matching the refractive index of sucrose solutions varied between 1*339-1.340. No significant trend was found in the distribution of refractive indices along the lens capsule. Hence the lens capsule with the adhering epithelial cells may be taken as a film with uniform refractive index. The refractive indices of the surface of the decapsulated bovine lens at different topographic positions were obtained by the minimum light scattering technique. In Fig. 2 the distribution of these refractive indices along the anterior part of the lens equator is given. Similar distribution along the posterior surface is expected except in a narrower range.
FIG. 2. Topographic distribution of refractive indices in bovine lenses (top view). The staggered line is the projected pathway of the laser beam through the lens. The bracketed numbers are the refractive indices obtained by direct measurements of the refractive index of different sections. The non-bracketed numbers are the refractive indices obtained by the minimal light scattering technique.
The reason for this statement is the fact that the nucleus has a higher refractive index than the cortex, (Philipson, 1969) thus the projected light path deviates from the straight line forming a hyperbole (Fig. 2 staggered line). The matching of the refractive index of the immersion liquid with that of the lens surface must be the average of the anterior and posterior surface refractive indices. The results indicate that the highest refractive index is in the middle and the lowest refractive indices are at the corners and that there is a smooth transition between the two extremes. Direct refractive index readings on selected sections of the cortex of bovine eyes show sufficiently good agreement with the data obtained from light scattering. As was expected, the nucleus has higher refractive indices than the cortex, and again shows a tendency of having higher refractive index at the center and lower at the corner of the lens nucleus. The same can be said of the refractive index change along the vertical axis of the lens (Fig. 3).
REFRACTIVE
FIG. 3. Topographic
distributim
INDICES
of rrfractive
IN
BOVINE
indices
ill hake
LENSES
lenses (frontal
view).
4. Discussion The primary role of the lens in the visual process is focusing the image on the retina. This is achieved by refraction. The refractive power is due (a) to the difference between the refractive indices at the interface and (b) to the curvature of the lens. The lens is embedded in front in the aqueous humor and posterior in the vitreous. Both have refractive indices of 1.336 (Kuck Jr., 1970). The average refractive index of the lens is given as 1.420 (Lerman, 1964). Our measurements indicate that the lens capsule almost matches the refractive index of its naturally-embedding media the vitreous and the aqueous. The average value of 1.335 found in the Abbe refractometer is probably somewhat low because of the incomplete coverage of the whole prism area. The values of 1.339-1.340 found by the immersion technique were quite reproducible and it showed that the lens capsule with the adhering epithelial cells is essentially uniform as far as refractive index is concerned. The results in Fig. 2 show that using as a probe, the minimum light scattering obtained in different non-interacting immersion media with different refractive indices, one is able to map the local refractive indices along the lens surface. The direct measurements of refractive index on cortical and nuclear sections indicate the same trend as that obtained by light scattering. These results are especially heartening considering the fact that in the direct measurements of the cortical slice not only the surface fiber cells contributed to the refractive index but a large number of fiber cells under the surface were also averaged in the measurements. The higher refractive index in the nucleus compared to that of the cortex has been known for some time (Philipson, 1969). The mapping of protein concentrations of the lens along the optic axis by microradiography showed a gradual increase in protein content from cortex to the center of the nucleus in all but the youngest (few days old) rat lenses. What this study has established is that in decapsulated bovine lenses there is a gradual and symmetrical change in refractive indices going from the center toward the corner along the horizontal axis as well as along the vertical axis. This means that the highest surface refractive index is at the position where there is the least curvature in the lens and vice versa. Since the refractive power of the lens originates both
356
I-‘. A. 1~ETTELHEIY
ANI)
T.
J.
Y.
%‘ANC,
from the curvature as well as from the refractive index difference at, the interface, it. seems that the adaptive process in the evolution tried to obtain a lens with more or less uniform refractive poffer along the whole organ; thus the increase in curvature was compensated for by t,he decrease in refractive index. Since the refract,ive index is due largely to the concentration of the crystallins in the fiber cells, our finding JVOUM imply that the highest concentration per unit volume of these proteins are in the central area of the lens within each fiber all along the surface. At the same time. however, we found previously that the width of the fiber cells is narrower along the anterior-posterior surface than at the corner (Bettelheim and Vinciguerra, 1971), SO it is quite possible that the concentration of crystallins along a unit length of a fiber cell is uniform throughout the lens and only the width of the fiber cell. i.e. its packing is changing. ACKNOWLEDGMENTS
This research was supported by a PHS grant 1J.S. Public Health Services.
EY-00501-(J4
of the National Eye Institute,
REFERENCES Bettelheim, F. A. and Vinciguerra, 11. J. (19il). Ann. N.I’. dead. 81%. 177,427. Bettelheim, F. A., Vinciguerra, M. J. and Kaplan, D. (1973). E.rp. Eye Res. 15, 149. Gilles, P. (1968). Double Liaison 152,45i. Ku& Jr., J. F. R. (1970). In Biochemistry qf the Eye (Ed. Graymore, C. K.). P. 190. Academic Press, New York. Lerman, S. (1964). Cataract. Thomas. Springfield, Ill. Philipson, B. (1969). Incest. Ophthdmol. 8, 3. Vinciguena. M. J. and Bet,telheim, F. A. (1971). Exp. Eye Res., 11, 214.