Original Articles Experimentally determined optical properties of goldfish cones and rods - IOVS
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Original Articles
Experimentally determined optical properties
of goldfish cones and rods
Frank L. Tobey, Jr.,0 Jay M. Enoch," and John H. Scandrett" °
The optical properties of goldfish photoreceptors were evaluated using the special microscope-
microspectrophotometer device. Mode patterns transmitted were identified for both natural
and reverse path illumination. Photarnechanical changes with adaptive state could be identified.
Complex mode interactive phenomena and cross-coupling of energy were observed. These are
related to the fact that there are double cones and apposition or near apposition of neighboring
receptors in this species. The directionality of individual cones and rods icas quantified by
making use of the principle of reversibility of light path. Radiation patterns were recorded
and analyzed on a computer controlled microdensitomctcr. Individual cones and rods were
shown to exhibit marked directionality. Light collection properties of cones were strikingly
evident.
Key words: goldfish, retina, photoreceptors, waveguide modal patterns, cross-coupling,
directionality, Stiles-Crawford elfect, light collection, photomechanical changes,
rods and cones.
.his laboratory has been concerned with which can be used to study the waveguide,
the optical properties of the retina and how directional, and spectral properties of in-
these affect the overall functioning of the dividual receptors and of small groups of
visual system. Recently, we described a receptors.1 A key feature of the instrument
special microscope-microspectrophotometer is the introduction of an auxiliary lens be-
yond the microscope objective giving ac-
cess both to an image of the specimen and
to an image of the objective back focal
From the Washington University School of Medi-
cine, Department of Ophthalmology, and The
plane (BFP). This permits improved iso-
Oscar Johnson Institute, 660 S. Euclid Ave., lation of that radiation which has tra-
St. Louis, Mo. 63110. versed the receptor, whether for identifica-
This research lias been supported in part by tion of radiation pattern or for estimation
National Eye Institute Research Grant No. of directivity or of spectral transmissivity.
EY 00204, National Institutes of Health, The instrument is designed to make use of
Bethesda, Md. 20014.
the waveguide-like behavior of the retinal
Submitted for publication May 20, 1974.
receptors.
"New address: Department of Ophthalmology,
University of Florida College of Medicine, To date, we have concentrated on study-
Gainesville, Fla. 32610. ing the waveguide behavior and directional
00
Department of Physics, Washington University, properties of receptors. Frog and rat rods
St. Louis, Mo. 63130. were shown to be markedly directional
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/20188 Tobeij, Enoch, and Scandrett Investigative Ophthalmology
January 1975
*>*•
Fig. 2. This is a picture of a goldfish double-cone
pair taken at the level of the cell nucleus. The
Fig. 1. This double cone clearly remains in appo- level roughly approximates the external limiting
sition vitread to the external limiting membrane membrane. At this level this cell pair is still in
(ELM). One cell nucleus (n) is penetrating the close apposition. The bar signifies 5 ^m (courtesy
ELM. This is a highly light-adapted specimen Professor A. I. Cohen, Washington University,
with the pigment epithelial fibrillar projections St. Louis).
and pigmented granules penetrating nearly to the
ELM. Note the very fine myoid projections to the
rods which are now deeply sheathed by the
pigment in the fibrils. The bar signifies 5 /mi. For observing cones, dark adaptation was
(Courtesy Professor A. I. Cohen, Washington followed by 30 to 40 minutes of light adaptation
University, St. Louis). in a small tank fitted with two 15-watt fluorescent
bulbs on one side and surrounded by diffuse white
reflectors. This period was chosen to produce at
optically (see Appendix II). 1 ' 2 In the pres- least a partial photomechanical contraction of the
ent work, these investigations have been cones without excessive return of pigment epithelial
pigment which renders the retina too opaque for
extended to cones—specifically goldfish
observation and difficult to separate during dis-
cones. We have also looked for optical section. Under these conditions we would expect
coupling between members of the double rods, which respond slowly, to be only partially
cones, which might be expected because extended toward their light-adapted photo-
of their close apposition. Finally, we com- mechanical state.3
pare optical directional effects of individual For observing rods, the light adaptation period
was omitted. Thus each type of receptor was ob-
rods and cones in the same species by re- served in a photomechanical state approximating
verse path illumination. its normal functional state." Since dissection took
place under white light, the photolabile pigments
Experimental were largely bleached.
Six- to eight-inch goldfish (Carassius auratus) The fish were decapitated and the anterior half
were dark-adapted for 18 to 22 hours. The long
dark-adaptation permits the retina to come away "In what follows "light adapted" refers to the partial
easily from the pigment epithelium during dissec- state of photomechanical light adaptation we were able to
achieve, Similarly, "dark adapted" implies photomechanical
tion. dark adaptation only.
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume 14 Optical properties of goldfish cones and rods 9
Number 1
Fig. 3. Electron micrograph of a section through a double cone at the level of the ellipsoid.
Cells are still in apposition. {Courtesy Dr. G. Marion Hope, University of Louisville.)
of the eye was cut away. We found that the able to observe the specimens; some preparations
vitreous adhered strongly to the retina, so that all were usable up to an hour before scattering be-
specimens consisted of a flat preparation of retina came severe. However, in earlier work with rabbit
with a relatively thick layer of attached vitreous. retinal choroidal blocks,4 silicone oil did not
The preparation was immersed in a 1.5 mm. deep prevent clouding for anywhere near the duration
chamber filled with silicone oil" and fitted with a obtained in goldfish. The success with this moun-
cover slip. In order to provide adequate working tant medium in this experiment suggests that its
distance for this deep chamber, the Zeiss Neofluar efficacy for the same purpose in warm-blooded
objective was replaced with a Leitz UMK 50 animals should be reappraised.
metallurgical objective. The specimen image was masked to isolate
The silicone oil encapsulates the preparation so receptors of interest and sequential photographs
that only natural aqueous fluids come in contact were taken of the specimen and BFP images in
with the tissue. This eliminates the problem of rapid succession. A calibrated density step wedge
selecting an artificial immersion medium, and some was recorded for each film. .
gaseous exchange is permitted. This procedure Most specimens were observed under reverse
greatly extended the period during which we were path illumination (receptors down in the cham-
ber) in order to determine the directionality of the
"Dow Corning 200, 50 Centistokes viscosity. Dow Coming receptor-waveguide to incident radiation.1' - If one
Corporation, Midland, Mich. can measure the angular distribution of light from
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201810 Tobey, Enoch, and Scandrett Investigative Ophthalmology
January 1975
Fig. 4. Electron micrographs of a section through the outer segments o£ a pair ot double cones.
Cells are now separate. The smaller cross-section outer segments and ellipsoid belong to sur-
rounding rods. Note that many of the rods are also effectively in apposition to each other
(Courtesy Dr. G. Marion Hope, University of Louisville).
the receptor acting as a radiator, one can infer Quantitative experiments were done with 575
the angular acceptance properties of the same nm. light on the special microscope. Qualitative
optical fiber (receptor) as a receiver on the basis observations were performed on a modified Leitz
of reversibility of light path.1- 2>4-6 The light microscope7 using a 533 nm. interference filter.
distribution in the back focal plane of the Cells evaluated for record were limited to fresh
microscope constitutes a map of the angular preparations where receptor orientation was good
distribution of light as it leaves the specimen relative to the optic axis of the microscope and
image plane. By masking, the contribution of a where the cone mosaic could be readily discerned.
single receptor can be isolated for study in the Records were photometered with a computer
back focal plane. For comparison with reverse controlled microdensitometer which converts film
path excitation conditions, a number of specimens density to log relative irradiance and allows the
were observed under natural path illumination. operator to take a profile along any chosen merid-
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume J4 Optical properties of goldfish cones and rods 11
Number 1
Fig. 5. Possible flux density patterns for two
identical touching rods. Taken from the paper
by W. Wijngaard,13 with his kind permission. Fig. 6. Light-adapted goldfish rod (r) and cone
(c) layer. The cones are contracted toward the
external limiting membrane (arrow at top of
ian through any point of a recorded pattern photograph). The rods are deeply sheathed in the
(Appendix I). Half-power widths were taken by pigment containing pigment epithelial fibrils. The
measuring profile width 0.3 log units below the rod myoids are highly extended. Note that nu-
peak of the profile. Widths were corrected for the merous rod receptors come into direct apposition
diffraction effect of the mask at the specimen in this adaptive state. This close association in-
image plane (Appendix II) and for the refractive fluences coupling of energy between receptors
index of the optical medium (assumed equal to and mode interaction. The bar signifies 5 (*m
1.334).2 (courtesy Professor A. I. Cohen, Washington Uni-
versity, St. Louis).
Results and Discussion
Double cones are readily distinguished
from rods in our preparations because of
their much greater brightness, both by
natural path illumination and by reverse
path. Single cones presented somewhat
more of a problem, especially by reverse
path, as their brightness level was less
than that of the double cones. Fortunately,
the double-cone receptors are arranged in
a well-defined rectangular mosaic in some
parts of the retina.8 The single cones regu-
larly occur near the center of the rectangles
defined by the double cones and displayed
much larger and more complex mode pat- Fig. 7. Goldfish cone (member of a double) ra-
diating a bilobed modal pattern at the specimen
terns than the rods. These differences, to- image plane. Reverse path illumination. Mask,
gether with the photomechanical effects, 30 /xm.
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201812 Tobey, Enoch, and Scandrett Investigative Ophthalmology
January 1975
Fig. 8. Near-field without mask, including the cone of Fig. 7. (See arrow. ]
enabled us to isolate the different receptor photomechanical effects reported in the
types for study. literature. The cones should respond first
Under reverse path illumination the cone with the onset of light adaptation by con-
mosaic appeared less prominent in the dark- tracting toward the vitreous. As the myoid
adapted samples than in the light-adapted contracts it increases in diameter, the mode
ones. In the dark-adapted samples, the lat- patterns become larger, and higher order
ter plane was reached before the rod mode patterns occur more readily. Simul-
mode-patterns were first detected. Cone taneously, the plane of maximum cross-
mode patterns also appear larger, and the section moves toward the vitreous. On the
more complex mode patterns seem to oc- other hand, the photomechanical response
cur more frequently in the light-adapted of the rods and of the pigment epithelium
retinas. occur more slowly for light adaptation.3
These observations are consistent with At the level of maximum brightness, the
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume J.4 Optical properties of goldfish cones and rods 13
Number 1
most common mode-pattern observed in
cones is the bilobe, but the dot and ring
or linear trilobe patterns were also fre-
quently observed. The more complex dot
and double ring occurred regularly. Oc-
casionally, still higher order mode pattterns
were detected. The lowest order single lobe
pattern was rare.51 For the rods, the bilobe
and annulus are overwhelmingly the most
common, but the dot and ring pattern oc-
curs regularly, as does the single lobe.
In light-adapted samples under reverse
path illumination we can follow the trace Fig. 9. Far-field radiation pattern corresponding
of the guided radiation over a considerable to Fig. 7. Added white marks are fiducial marks
range in the double cones by starting with for the computer.
the plane of focus well toward the vitreous
from the receptor cells and then moving Table I. Half-angles* at 0.3 log unit below
slowly toward the sclera. One first en- peak irradiance for goldfish receptors
counters two very small, weak, separated transmitting bilobe modes under reverse
mode patterns of low order (single lobe path illumination
or bilobe). The two patterns enlarge and
intensify progressively, sometimes forming Cones \ Rods
higher order patterns which may be dif- 2.4° (Figs. 8 and 11) 2.751
2.2 0.9J (Fig. 13)
ferent in the two cells. Beyond the point 2.5
3.21
where the patterns are in near contact, 1.2} 2.3
four types of behavior may occur: (1) the 2.8
2.3
patterns proceed parallel to each other; "These iire defined hy the angle between the axis and a
some interaction between the patterns may generatrix of the light cone with vertex nt a point of
energy radiation near the termination of the reccptor-
be apparent, but not always, (2) the pat- w;ivej{uidi_'.
t Average of two separate records nn the same cell.
terns may merge to form a larger pattern, JOne lobe only. Weaker lobe fell below the the half-
sometimes of higher order (dot and ring irradiancL1 level.
or dot and double ring). Details of the
central part of the pattern often become the traces can be observed to separate
indistinct, (3) an abrupt jump of radia- slightly and form smaller, lower order mode
tion may occur from one guide to the other, patterns which eventually disappear, not
and (4) occasionally the radiation jumps necessarily at the same level. The preced-
to what appears to be a different wave- ing observations seem to be in accord with
guide than the two we have been observ- electron micrographic studies of goldfish
ing. From our observations, we cannot judge double-cone morphology. Fig. 1 shows a
whether this third guide is a cone or a goldfish double cone in section parallel to
rod. If the third guide is a rod, it is tem- the receptor axes. Note the extended re-
porarily supporting a much higher radia- gion of close apposition including the
tion density than we ever observed in the nuclear and part of ellipsoid levels. Figs.
bulk of the rods. More than one of the 2 through 4 are a series of sections through
above phenomena may occur in the same double cones approximately perpendicular
pair of guides at different levels. It is over to the receptor axes at different levels (dif-
this range of levels that the mode patterns ferent samples). The close apposition at
are most intense. the nuclear and part of the ellipsoidal
Proceeding further along the double levels is evident (Appendix III). In this
cones, the patterns decrease in intensity, region, strong interaction between the elec-
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201814 Tobey, Enoch, and Scandrett Investigative Ophthalmology
January 1975
Fig. 10. Contour map of the pattern in Fig. 9, generated by the computer-controlled scanning
densitometer. The brighter points delineate the traverse along which a profile was taken
in Fig. 11.
Fig. 11. Profile along the traverse shown in Fig. 10. Ordinate: log relative irradiance xlOO.
Abscissa: distance along the traverse across the film frame; also proportional to angle at the
specimen plane (uncorrected). See Table I.
tromagnetic radiation traversing the two havior is already occurring in the axons
guides would seem likely. The observed vitread to the point where the double cones
separation of the mode patterns scleral to come into apposition, and well within the
the region of interaction also fits with mor- outer nuclear layer. This requires that the
phologic separation of the double-cone guide must be surrounded by a medium
outer segments. of lower refractive index. Since our prep-
The small, separated mode patterns in- arations were only minutes old and since
itially observed show that waveguide be- no foreign fluids were added to the tissue
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume 14 Optical properties of goldfish cones and rods 15
Number 1
Fig. 14. Goldfish cone radiating a linear trilobe
modal pattern at the specimen image plane.
Reverse path illumination. Mask 35 j«m.
Fig. 12. Goldfish rod radiating a bilobed modal
pattern at the specimen image plane. Reverse
path illumination. Mask 20 /tm.
Fig. 15. Far-field pattern corresponding to Fig. 14.
Table II. Half-angles at 0.3 log unit below
peak irradiance for trilobe or dot and
ring patterns radiated by goldfish cones
under reverse path illumination
A B C
Symmetric Ast/mmetric Inferred
2.7° (Figs. 15 2.3° 3.8°
and 16)
2,7 2.2 3.9
2S 3.4
I"). I',ir ill I d i a d j . i t ji i l l Matlci'ii i n r r i -,rii 2.7 4.2
Fig. 13. Far-field radiation pattern corresponding 2.2 3.5
to Fig. 12. The pattern has been rotated in 2.0 (Figs. IS 3.1
printing. and 19)
(silicone oil is not miscible with tissue optimal plane of focus in the retina. Eber-
fluids), a comparable situation may exist le"' has suggested that differences in cone
in life so that some energy is coupling into lengths provide a correction for chromatic
these guides vitread to the external limit- aberration. However, it is the level of the
ing membrane. This need not be the plane input of the waveguide which is pertinent
of maximal coupling of energy into the re- to the optimum plane of focus rather than
ceptor waveguide, however. The plane of the position of the outer segments.
maximum coupling is, by inference, the The frequent occurrence of power trans-
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201816 Tobey, Enoch, and Scandrett Investigative Ophthalmology
January 1975
Fig. 16. Profile of Fig. 15. Coordinates as for Fig. 11. See Table II.
Table III. Half-angles measured at 0.3
log unit below peak irradiance for mode
patterns radiated by goldfish cones under
natural path illumination
Single lobe Bilobe Dot and ring
1.6° 2.6° 3.4°
1.5 3.0
fer between double cones indicates that,
in this species at least, through-the-retina
microspectrophotometry has to be per-
Fig. 17. Goldfish cone radiating a dot and ring formed by natural illumination path, since
modal pattern at the specimen image plane. Re-
verse path illumination. Mask 35 p-m. by reverse path light emerging from one
receptor may have traversed another re-
ceptor outer segment. This is less likely
to be a problem by natural path because
of the separation of the outer segments.
For species such as the turtle where one of
the double cones contains a pigmented oil
droplet, its effect must be considered for
both light paths.
Double cones could also be followed for
some distance under natural path illumina-
tion. Focusing from the pigment epithelial
side toward the vitreous, cone mode pat-
terns were initially separated, then ap-
proached each other at the largest cross-
Fig. 18. Far-field pattern corresponding to Fig. 17. section. Cross coupling and interaction
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume M Optical properties of goldfish cones and rods 17
Number 1
Fig. 19. Profile of Fig. 18. Coordinates as for Fig. 11. Note asymmetry of irradiance profile.
See Table II.
were less prominent than by reverse path.
We could not trace the pattern much be-
yond the level of maximum cross-section as
it then became indistinct. This again agrees
well with the morphology previously de-
scribed. Photomechanical changes observed
were consistent with the literature. Most
frequent mode patterns near cone termina-
tions were bilobes, with single and trilobe
(dot and ring) patterns being common.
As in most other vertebrates, the superior
light collection properties of cones was
most striking.1' 7- ""'* Mode patterns ob- Fig. 20. Pair of goldfish double cones each ra-
diating a dot and ring at the specimen image.
served in rods by natural path appeared Some interaction appears to be occurring. Re-
similar to those observed by reverse path. verse path. 48 (tm mask.
A phenomenon which was frequently ob-
served and which may also indicate that
radiation interaction takes place between
receptor waveguides was the occurrence of
an "interference-like" pattern of parallel
wavy lines in the region between the cones
occupied by the rods. This occurred both
with reverse path and natural illumination
and in both the dark- and light-adapted
specimens. Close examination showed that
this appearance resulted from the small,
predominantly bilobed modal patterns
transmitted by the rods having their nodal
lines (the line of symmetry separating the Fig. 21. Far-field pattern corresponding to Fig. 20.
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201818 Tobey, Enoch, ami Scandrett Investigative Ophthalmology
January 1975
Fig, 22, Density profile of a step-wedge pattern used to calibrate film for irradiance.
two lobes) aligned nearly parallel to each pattern under reverse path illumination.
other over a region including perhaps ten Mask size was 30 ^m. Fig. 8 shows the
to a hundred or more rods. This is highly corresponding far-field pattern. Fig. 9
suggestive of the results of Wijngaard's14 shows the specimen image plane without
theoretical treatment of guided modes in a mask taken about 90 seconds after Fig.
two circular dielectric waveguides (Fig. 7. The cell is readily identified. Fig. 10
5). In fact, this figure depicts flux density shows a contour map produced by the
patterns which would be remarkably simi- computer controlled microdensitometric
lar to what we observe if they were ex- scanner from the pattern shown in Fig 8.
tended to a large group of similar wave- The bright spots identify the traverse
guides. Mclntyre and Snyder1" have car- along which the profile was taken. In the
ried out a similar analysis. profile shown in Fig. 11, the ordinate is
Inspection of Fig. 6, which is an electron log relative irradiance and the abscissa
micrograph of a transverse section con- measures displacement along the traverse
taining a group of fully light-adapted gold- across the pattern. It is also a measure of
fish rods, suggests that the conditions for the angular distribution of radiated energy
interaction may be fulfilled: the rods are far-field plane, because of the properties
closely spaced along a considerable part of the back focal plane of the objective.
of their length (see also Fig. 4). Scanner operation is covered in more de-
It should be emphasized that comparable tail in Appendix I.
cross-coupling and mode interactive effects Figs. 12 and 13 show a bilobe pattern
have not been observed in mammalian radiated by a goldfish rod at the specimen
retinas (human, rabbit, monkey, and rat). image and far-field planes, respectively. Il-
The high resolution observed in these lumination was by reverse path and a 20
retinas acting as fiber optics bundles makes pm mask was used.
it unlikely that marked cross-coupling ef- Table I presents the results of a series
fects occur in these species.4' 1C'1S of measurements of the radiating half-
Fig. 7 shows the specimen image plane angles measured at the half-irradiance
of a goldfish cone radiating a bilobe mode point 0.3 log units below peak) for indi-
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume 14 Optical properties of goldfish cones and rods 19
Sumber J
Fig. 23. Fragmented goldfish double cone sus- Fig. 24. Interference pattern superimposed on
pended in aqueous humor of the same species. the .same two cell fragments. Note that receptor
The picture was taken with a Zei.ss Jamin-LebedefF inner and outer segment components differentially
Interference Microscope with the interference com- displace the fringes, indicating differences in index
ponent removed. Note that the two fractured of refraction between outer segment and ellipsoid.-"
receptors remain in apposition, suggesting that
there may be adhesion between the double cones.
vidual goldfish rods and cones. Illumina- "If one observes the terminations of well-
tion was by reverse path in all cases and oriented rods or cones, one notes significant
the values have been corrected for mask changes in transmission of waveguide mode
diffraction and for the refractive indexes patterns (and also changes in patterns)
of the optical medium (1.334). The anom- when the angle of incidence is changed by
alous value appearing in each column only 1°, 2°, or 3°. The magnitudes for rods
represents, in each case, a highly asym- and cones are not quite the same, but the
metric bilobe in which the weaker lobe differences are limited. Thus, individual re-
fell more than 0.3 log units below the peak. ceptor directivity, determined by the study
Based on the reciprocity principle of Helm- of mode pattern transmission characteris-
holtz, these should also represent the rela- tics, is apparently far greater than that
tive ability of the cell to accept radiant found in psychophysical determinations of
energy as a function of angle of incidence the Stiles-Crawford effect of the first kind."
at the cell. Table I implies that individual The present quantitative work using a
cones and rods are optically highly direc- different technique shows that in the gold-
tional. Differences in directionality between fish essentially comparable results are
radiation patterns isolated from rods and found. Compare these results also with the
cones were small. Given the size of sample corrected rat rod data of Table IV.
and biases in selection of cells for evalua- Our measured half-angles are small rela-
tion, it is impossible to state at present tive to those observed for both the cone
whether or not systematic differences exist. and the rod Stiles-Crawford (S-C) effect
It is instructive to compare our present found for a recently studied human blue-
results with other measurements of direc- cone monochromat.111 The half-angle found
tional properties of vertebrate visual sys- for the human scotopic Stiles-Crawford
tems. In earlier studies in this laboratory, function was 8.5° and for the photopic
albino rat, squirrel monkey, rhesus monkey, S-C function 4.8°. The rod function was
and human retinas were examined under assumed to be symmetric for this purpose.
natural path illumination.3 Light trans- The photopic value was obtained by fit-
mitted to receptor terminations was ob- ting Stiles' 1937 equation20
served as the angle of incidence at the
= 0.08)
retina was varied. The results were sum-
marized as follows: to the experimental data.
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201820 Tohei/, Enoch, and Scandrctt Ophthalmology
January 1975
Here \\ is relative visual sensitivity and r level. Column B refers to six cases where
is displacement in millimeters across the one side was below the half-power point.
entrance pupil of the eye, measured from Column C refers to the same data as Col-
the position of the S-C maximum. One umn B but the widths of corresponding
millimeter in the entrance pupil of the eye "complete" patterns were estimated by
is equivalent to 2.5° change in angle of taking the angle between the center of the
incidence at the retina. dot and the half-power level of the stronger
Raynauld-1 has measured photopic and side and doubling the value. Column C
scotopic Stiles-Crawford functions for gold- does not agree well with Column A. On
fish by recording spiking rates from single the other hand, it is interesting to note that
ganglion cells while varying the input Column B falls very close to the cone bi-
angle from a photostimulator. The illum- lobe values of Table I and even Column
inated field, 1.0 mm. in diameter, was A is quite close to the latter.
slightly smaller than the center of the Fig. 20 shows both members of a double
ganglion cell receptive field, so that a large cone each radiating a dot and ring pattern
number of receptors were stimulated. He and showing evidence of interaction. Fig.
also measured the stimulus response curves 21 is the corresponding far-field pattern.
for the same ganglion cell under normal The fact that the patterns do not coincide
incidence, so that the angular and gain exactly shows that the cones were radiat-
components of sensitivity could be deter- ing in slightly different directions.
mined. Measurements were made by both For completeness, we include in Table
natural and reverse path, but the former III some values of far-field radiation pat-
is the relevant measurement for compari- terns measured on goldfish cones by natural
son with data considered here. Without path illumination. The different mode pat-
question, the half-angle of the single pat- terns present are identified. The data of
terns analyzed here were quite a bit smaller Table III bear no relation to the acceptance
than the half-angles of the directional sen- angle of the cone receptor waveguide in
sitivity functions reported by Raynauld the light-adapted eye, since under these
and Laviolette.-1' conditions the normal input of the guide
We could not analyze groups of cells in is at the other end. They are of significance
this work because of the inhomogeneity when considering tapetal back reflectance.
of the receptor population in goldfish.
We have also taken measurements on
We gratefully acknowledge the skillful help of
modal patterns of the next higher order
Professor C. M. Hope and Mr. Marian Taylor
(dot and ring and linear trilobe) radiated of the Department of Ophthalmology, University
by goldfish cones under the reverse path of Louisville School of Medicine, and Professor
illumination. Figs. 14 and 15 show a linear A. I. Cohen and Miss Shirley Freeman of the
trilobe in the near- and far-field, respec- Department of Ophthalmology, Washington Uni-
versity School of Medicine, in preparing the
tively. Fig. 16 shows a profile. Interpreta- electron micrographs. The assistance of Miss Susan
tion of these measurements is uncertain Messenger and Mrs. Beverly Lawrence was greatly
because the patterns are nearly always in- appreciated, as was the enthusiasm of students
complete or highly asymmetric. Figs. 17 |ohn Boles, Tom Deutsch, and Boh Langdon, who
developed the dissecting skill essential to this work.
and 18 show the near- and far-field pat-
Our thanks also to Ed Donegal) for his help with
terns for a relatively complete dot and photographic reproductions.
ring. Fig. 19 shows the profile, with one
side of the ring falling below the half-
power point. These last results are col- REFERENCES
lected in Table II. Column A refers to two 1. Enoch, |. M., and Tobey, lr. L.: A special
cases where both sides of the ring (or microscope-microspectrophotometer: optical
both side lobes) fell above the half-power design and application to the determination
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume 14 Optical properties of goldfish cones and, rods 21
Number 1
of waveguide properties of frog rods, J. Opt. monochromatic rays entering the eye pupil at
Soc. Am."(i3: 1345, 1973. dilfeient points and a new colour ell'cct, Proc.
2. Tobey, F. L., and Enoch, J. VI.: Directionality Roy. Soc. Loncl. Ser. B 123: 90, 1937.
and waveguide properties of optically isolated 21. Raynauld, f. P. ( Departement cle physiologic,
rat rods, INVEST. O P H T H A L M O L . 12: 873, 1973. Universitc de Montreal): Personal communica-
3. Ali, M. A.: Les reponses retinoinotrices: tion. This author also calls attention to |ohn
caracteres et inecanismes, Vision Res. 11: Laviolctte, "Stiles-Crawford Ellect and Cain
1225, 1971. of Photoreceptors in tJie Isolated Goldfish
4. Enoch, |. M., and Glismann, L. E.: Physical Retina, " Thesis, University cle Montreal, 1974.
and optical changes in excised retinal tissue: 22. Clickstein, VI., Labossiere, E., and Yager, D.:
resolution ot retinal receptors as a filler optics Cone position in the partially light-adapted
bundle, INVEST. OPHTI-LALMOL. 5: 208, 1966. goldfish retina, Ajiat. Rec. IG3: 189, 1969.
5. Enoch, J. M.: In: Fiber Optics, Knpany, N. S., 23. Thorpe, S. A.: The Ellect of Chromatic
editor. New York, 1967, Academic Press, p. Adptation ami Temperature on the Spectral
382. Sensitivity of the Goldfish, Carassius auiattis,
6. Enoch, J. VI., and Scandrett, J.: Human Ph.D. Dissertation, Brown University, 1972.
foveal far-field radiation pattern, INVEST. 24. Harosi, F. S.: Frog Rhodopsin in situ:
OPHTHALMOL. 10: 167, 1971. Orientational and Spectral Changes in the
7. Enoch, |. VI.: Optical properties of retinal Chromophores of Isolated Retinal Rod Cells,
receptors, J. Opt. Soc. Am. 5 3 : 7 1 , 1963. Ph.D. Dissertation, Johns Hopkins University,
8. Engstrom, K.: Cone types and cone arrange- 1971.
ments in the retina of some ci/prinkls, Acta 25. Cohen, A. I. (Department of Ophthalmology,
Zool. 4 1 : 277, 1960. Washington University, St. Louis): Personal
9. A description ol the various modal patterns communication.
and a summary of their technical designations 26. Enoch, |. VI., Scandrett, J., and Tobey, F. L.:
is given by: Enoch, [. VI.: Visualization of A study of the ellects of bleaching on the
waveguide modes in Retinal Receptors, Am. width and index of refraction of frog rod
|. Ophthalmol. 5 1 : 1107/235, 1961. outer segments, Vision Res. 13: 171, "l973.
10. Ebcrle, II.: Cone length and chromatic aber-
ration in the eye of Lchistcs reticulatus, Z. Appendix I
Vergl. Physiol. 57: 172, 1967.
11. Tanslcy, K., and |ohnson, B. K.: The cones Data reduction. The radiation patterns of indi-
ol the glass snake's eye, Nature 178: 1285, vidual retinal cells were analyzed using a computer-
1956. controlled film scanner and an interactive computer
12. Enoch, |. VI.: Nature of transmission ol program which allowed an operator to inspect the
energy in the retinal receptors, |. Opt. Soc. image I mm a radiation pattern, then plot a log-
Am. 5 1 : 1122, 1961. relative irradiance profile along a selected scan
13. Winston, R., and Enoch, ). VI.: The retinal line through the observed image.
cone as an ideal light collector, |. Opt. Soc. The measuring apparatus consisted of a com-
Am. (II: 1120, 1971. puter-controlled microdensitometer and display
14. Wijngaard, W.: d u e l e d normal modes of two system. A PDP 11 computer drove a cathode ray
parallel circular dielectric rods, |. Opt. Soc. tube as a programmable point source of light
Am. 3: 944, 1973. which was imaged on the 35 mm. film. Each film
15. Vlclntyie, P. D., and Snyder, A. W.: Power strip was entered in a film library accessed by a
transfer between optical fibers, |. Opt. S o c high-speed film advance system under computer
Am. (»:$: 1518, 1973. control at the measurement platen. A double photo-
16. O h / u , II., Enoch, J. M., and O'llair, J.: multiplier system compared transmitted light
Optical modulation by the isolated retina and through a computer-addressed point with the
retinal receptors, Vision Res. 12: 231, 1972. source intensity on the CRT face. The transmitted
17. O h / u , II., and Enoch, |. VI.: Optical modula- light was integrated up to a constant photon
tion by the isolated human lovea, Vision Res. count; the logarithm of the transmitted-to-source
12: 245, 1972. ratio was then electronically produced and
18. Enoch, |. VI., and Mope, C. VI.: Diiectional digitized on a 0 to 1,023 scale called scanner
sensitivity ol the loveal and parafovcal retina, density. This constant noise system took 20 to
INVEST. OPHTMALMOL. 12: 479, 1973. 1,000 microseconds per point, depending on den-
19. Daw, N., and Enoch, J. VI.: Contrast sen- sity.
sitivity, Westheimer function and Stiles-Craw- The CRT face was addressed on a 4,096 >: 4,096
lord ellect in a blue-cone nionochiomat, point grid, and the CRT spot size approximately
Vision Res. 13: 1669, 1973. equalled the least-count spot displacement. The
20. Stiles, W. S.: The luminous elficieney ol density measuring system was calibrated so the
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/201822 Tobeij, Enoch, and Scandrett Investigative Ophthalmology
January 1975
full numerical density range 0 to 1,023 cor- Appendix II
responded approximately to two decades of Correction of measured light-cone angles for
density. diffraction at the masking aperture. We have
Scanned images were displayed in discrete point modified our procedure for correcting observed
form on a Tektronix 611 storage display system light-cone angles for the diffraction spreading
under the control of an operator. A sequence of introduced by the small masks at the specimen
Fortran programs was written to display selec- image plane. Our previous approach was described
tively magnified sections of the images using a on p. 875 in Reference 2. A subtractive correction
crude density contouring technique. Relative to was obtained from an empirical correction curve
the displayed density pattern under measurement, of the form
the operator selected the most representative pro-
file through the pattern by using a family of
a, = a,, - —
computer-interrogated potentiometers. A pair of d
knobs controlled the coordinates of the center of
a displayed scan line, and a third knob controlled where «', was the corrected angular separation,
the aziimithnl angle. The length of the scan line 'i'o the observed separation, and cl the diameter
had been previously typed in at the computer of the mask aperture, k was determined empirical-
console (usually approximately \{\ of the frame ly by measuring the angular width of the BFP
length). Fig. 10 shows the selected scan line as pattern from an illuminated single glass fiber
a superposition of spots on the magnified display. with different sized apertures at the specimen
image plane (Fig. 3 of reference 2).
A 256 point scan was taken in scanner density
units, which measure approximately the logarithmic Further work has convinced us that this cor-
film density of the retinal pattern. These values rection procedure suffers from serious defects.
were converted to log-relative irradiance (LRI) It requires the same subtractive correction for
by interpolating in a table of scanner density the angular separation of any two structural fea-
versus LRI, then plotted as shown in Fig. 11. tures in a BFP record, whether their separation
is large or small. It can easily lead to negative
The conversion table was separately determined
cone angles, a nonphysical result.
for each experimental run by including an image
of a Kodak step-wedge, taken with the same A more logical approacli is to apply a multi-
optical system immediately afterward. The LRI plicative correction factor derived from the same
reaching the camera focal plane was directly type of glass-Fiber data and n curve completely
measured with a logarithmic light-meter probe analogous to the referenced figure:
positioned and read behind each step of the step- n,,.
wedge-image at the camera focal plane.
The conversion table relating scanner density
to LRI was separately determined for each experi- where aui, is the observed angular diameter of the
mental run by scanning the step-wedge image on glass fiber pattern for the same mask size, and
each film strip. Fig. 22 shows a profile through the «•„ the angular diameter of the glass fiber pattern
step-wedge, with scanner density coordinates on obtained by extrapolating to an infinite aperture
the vertical axis. A computer file was created (zero on the abscissa). This treatment is equiva-
holding a table of scanner density differences lent to assuming that each mask simply produces
above background versus measured LRI with one a magnification-like effect on the BFP image.
entry tor each discernible step. A linear inter- This agrees qualitatively with our visual impres-
polation program converted the 256 scanner density sions and the problems associated with the pre-
points to the LRI values, giving the resulting vious approach are eliminated (see Reference 2,
plot of Fig. 11. Fig. 4). Because 200 MM at the specimen image
Thus each strip of film had a direct calibra- approximately defined the illuminated region, larg-
tion relating log-relative irradiance to scanner er masks were set equal to 200 /xM for puqjoses
density, regardless of possible shift in gray level of this correction. Obviously, it would be desir-
or gamma of the processed film and regardless able to provide a more rigorous analytic treatment,
of possible shifts in numerical level and gain but none is available at present.
of the computer-controlled densitometer. Numerical results published in References 1
Finally, the spatial width on film of a par- and 2 require correction for this change in pro-
ticular cell pattern (which for a far-field image cedure. These corrections are listed in Table IV.
is proportional to angular width of the radiation In general, cone hall-angles of receptors are found
pattern) was inferred from the resulting graph to be much narrower than previously supposed.
(Fig. 11) by measuring the intercept width of a This has considerable significance in tenns of
line drawn 0.3 log unit down from the observed our understanding of the directivity of the single
peak. receptor and the Stiles-Crawford effect. This
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018Volume 14 Optical properties of goldfish cones and rods 23
Numhvr 1
Table IV. Corrected half-angles for rat rods and frog red rods. Illumination by
reverse path
Page Col, Line Fig. Description Old value New Value
Reference 2 (rat rods):
877 1 29 6 1 cell 2° 1.6°
877 1 32 5B 1 cell 8° 2.5°
877 2 6 8B —' 75 cells 17° 11°
878° 1 18, 19 — Approximately Mean 18° Mean 12°
100 cells Range 3° Range 1.7°
Reference 1 (frog red rods):
1,354 2 10 23 1 cell 5° 2.9°
1,354 2 13 25 1 cell 9° 4.4°
1,354 2 52 35 7 cells 7° 4.6°
1,354 2 51 36 — 100 cells 10° 6.3°
°Six records.
question will be treated in a subsequent publica- Labossiere, and Yager1-"-' and Thorpe-"1 is theo-
tion. The correction for the refractive index of retically possible for the double cones as well
the ocular medium (n = 1.334) is made just as for single cones. If adhesion is present, inde-
as before by applying Snell's law. pendent photomechanical action becomes ques-
tionable. There is no obvious specialized anatomic
Appendix III mechanism for adhesion observable (Fig. 2).'-'•''
On the other hand, Fig. 21 shows a double
An important question with regard to the cone
goldfish cone fractured in the myoid region of
pair is whether or not apposition also involves
the cells. The fact that this cell pair remained
some degree of adhesion. Glickstein, Labossiere,
in apposition after fracture suggests that the
and Yager1-- and Thorpe'-' suggested the possi-
cone pair may in fact be adherent in the myoid
bility of individual cone groups being subject to
region. It is in the myoid region that photo-
essentially independent photomechanical adaptive
mechanical changes take place. In Fig. 22, this
change. For example, red cones (only) might
same pair is seen by Jamin-LebedelT type (Zeiss)
contract toward the external limiting membrane
interference microscopy. The index of refraction
when the retina is irradiated with long wavelength
differences between the outer segment and ellip-
light. It has been shown that goldfish double
soid are demonstrated by the difference in fringe
cones are red-green receptor pairs.1-'1 Without ad-
shifts in the two parts of the cell.
hesion, the proposition suggested by Clickstein,
Downloaded From: https://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933059/ on 09/23/2018You can also read
