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A study of the effects of bleaching on the width and index of refraction of frog rod outer segments
A STUDY OF THE EFFECTS OF BLEACHING ON THE WIDTH AND INDEX OF REFRACTION OF FROG ROD OUTER SEGMENTS’ JAY M. ENOCH,~JOHNSCANDREI-T~ and FRANK L. TOBEY, JR.’ ’ Department of Ophthalmology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, U.S.A. and J Department of Physics, Washington University, Skinker and Millbrook Boulevards, St. Louis, Missouri 63130, U.S.A. (Reeeiwd 27 Jury 1971; in reoised form 31 March 1972)
INTRODUCTION RETINALreceptor cells behave like dielectric waveguides for electromagnetic radiation at optical wavelengths (e.g. ENOCH, 1967a, KRAUSKOPF,1966). A waveguide operating at wavelengths in a range comparable to its diameter does not transmit all WaveIengths unifo~ly, Hence an effective spectral absorption curve taken end-on though a receptor may
differ in detail from the absorption of isolated pigment, even allowing for pigment dichroism (e.g. ENOCH,1961). At a wavelength near a cutoff transition of a modal pattern, a cylindrical waveguide is critically sensitive to diameter and to the diference between the indexes of refraction of the core and the exterior medium, especialiy if the difference is smalf, as is usually the case for biological material. It becomes important to know if changes induced in the receptors, either in vitro or in vim, can produce changes in their waveguide behaviour. For example, this can be a significant question in through-the-retina microspectrophotometric studies. In the work described here, we have measured the widths of rod outer segments and their index of refraction along a path perpendicular to the long axis before and after bleaching. Isolated frog rods (Rana pipiens) with attached ellipsoid (fractured in the inner segment) were examined with a Zeiss interference microscope modified to function in the infra-red. All measurements were made at h = 826 nm in the infra-red in order to prevent bleaching by the measuring beam. F. von Hornbostel, (cited by KUHNE, 1877), first suggested that an increase in frog receptor diameter might be caused by the bleaching of the photolabile pigments. WOLKEN (1963) also reported a similar result. More recentIy FRIEDMANand KUWUBARA(1968) and KUWUBARA(1970, and personal co~unication) provided electron microscopic evidence of swelling and other physical changes induced by the action of light occurring in photoreceptors of rats and monkeys. WEALE(1970a, b) has studied the birefringence of frog retinal receptors and deduced from his results that changes inthe length of the receptor occur following bleaching. More recentiy WEALE(197Ia, b, c, d) has obtained data on frog rod birefringence and has provided an analysis of the components contributing to birefringence in the dark adapted and bleached states. He observed differences in index of refraction in the I This work has been supported in part by Research Grant No. Ey 00204 and by Career Development Award No. K3 15138 (to J.M.E.) of the National Eye Institute, and by Research Grant NO. RR 0396 of the Division of Research Resources (to -IS.), National Institutes of Health, Bethesda, Maryland. 171
111
JAY Xl.
ENOCH, Jon
SCAXDRETT AND FRASK L. TOBEY, JR.
third and fourth decimal place. SNYDER and RICHAIOND (1972) present a theoretical treatment of the effect of anomalous dispersion on receptor index of refraction and photolabile pigment absorption. Many authors in recent years have dealt with physical and chemical changes occurring in the outer segment due to the action of light. METHOD Apparatus A Zeiss interference microscope (Standard Universal [POL] Jamin-Lebedeff type) was adapted for use in the infra-red. A 150-W Osram Xenon arc was substituted for the standard Zeiss Mercury arc. A RairdAtomic infra-red interference filter (passband of 820-832 nm) was used at the field stop to provide a monochromatic beam. A calcite polarizer in a three-point mount replaced the subcondenser Polaroid in order to improve polarization and transmittance in the infra-red. Since the polarizer and interference filter were removed during bleaching, the three point mounting allowed rapid relocation in the dark. The quarter wave plate was replaced by one designed for X = 826 nm. An analyzer eyepiece (Zeiss No. G41-452 with special Wollaston prism elements) was used to introduce fringes in the field (Fig. 1). The 40x condenser and objective units were retained. The resultant field was imaged by a photoeyepiece onto the photocathode of an RCA 6914-A infra-red image converter fitted with an Et-Se eyepiece to magnify the image on the phosphor screen. An Exacta camera was used to record the phosphor display. The time of each photograph and of bleach duration were recorded on a constant speed penwriter. A Leitz stage micrometer provided dimensional calibration. The E-vector of the measuring light was set perpendicular to the long axis of the outer segment and parallel to the fringe pattern. Thus the E-vector lay in the plane containing the absorption axes of the visual pigment, but not in the absorption plane of certain photoproducts (e.g. HAROSI,1971). Sample preparation
Since it was expected that only small changes in size and optical path-length might result from bleaching, frog rods were used because of iheir size. Considering the resolving power of the light microscope at X = 826 nm, our ability to detect small changes in a mammalian receptor having a diameter of 1.5-2.0 p appeared questionable. Frogs were dark-adapted overnight. Time recording began with decapitation of the frog, after which a single razor slice served to remove the anterior half of the eye. The retina was removed with iris forceps and lightly dipped in the immersion medium to detach a small number of receptor elements. Only outer segments with attached ellipsoids were used, and measurements were limited to the outer segment. All dissection was conducted in total darkness with the aid of a second infra-red image converter and infra-red sources. Every effort was made to diminish the elapsed time between dissection and measurement in order to minimize artifacts in these preparations. Initially, aqueous humor from the second eye of the frog was used as the immersion medium for the rod preparation. Care was used to prevent evaporation of samples by storing them in closed chambers slightly larger than the drop volume. When it proved ditTicult to obtain sufficient aqueous from the second eye, fresh aqueous humor from the eye of a large goldfish was substituted without apparently altering the results significantly. Since tricaine solution was used to anesthetize the goldfish, it was necessary to carefully blot the eye in order to prevent contamination. The index of refraction of the immersion medium was measured with a temperature controlled Bausch and Lomb Precision Refractometer held at the average room temperature of 22’C. Our measurements of triple distilled water taken under standard conditions (589.3 nm, 20°C) matched standard values well. Significant values are listed in Table 1. All index values measured at h = 589.3 nm were corrected to the test wavelength of 826 nm (WASHBURSE, 1930). This correction assumes that the dispersion of the index for aqueous follows that of water. This is reasonable, since the index at 589.3 nm is close to that of water, and both fluids are transparent over the relevant wavelength range. Experimerltal procedure
Ten exposures of the interference pattern were taken in rapid sequence, followed by a IO-see bleach. Ten more exposures were taken immediately after the bleach. The aperture of the condenser was not altered during the bleach. Since the white bleaching light was observed to grossly bleach a fresh retina placed on the stage, and since thcrmopile (Eppley) measurements indicated that the energy/unit area delivered to the specimen should have been adequate to bleach all rhodopsin, bleaching was assumed to be complete. Sample heating is not a serious problem with this instrument since standard microscope optics filter out radiation of h > cu. 900 nm. As a check, temperature rise during a bleach was measured with a Yellow Springs recording microprobe thermistor placed in the light path on the microscope stage. Less than 1°C
173
Bleaching Efkct on Frog Outer Segment
TABLE 1. 1. Mean
INDEXOF REFRACTION
of values measured at 589.3 nm Aqueous humor
Temperature (“C)
Pure Hz0
Frog
Goldfish
Tricaine solution
20 21 22 23 24
l-33307 i-33301 1.33290 1-33282 1.33277
1.33609 l-33601 1‘335% 1.33587 1.33582
I.33499 l-33497 1.33486 1.33477 1.33466
1.33323 I.33318 l-33307 1.33296
---_ 2. Refractive index, no, of pure water at A = 826 nm source: International Critical Tables (1930) x n0 589.3 826
I.33300 1.32780
t
20°C 20°C
3. Index of refraction of measured specimensL xl adjusted n, measured (t = 22°C; II = 589.3 nm) (r = 22°C; X = 826 nm) Test day _. __--- ____ .._ 55 1.33472 1.32951 ii
1.33522 l-33527
1*33ooo 1.33061
72
1.33467
1.32945
r Values for fresh gold&h aqueous humor sample used with given frog retinal specimens on specific test days.
change in temperature was recorded in the plane of the specimen during a IO-se-cexposure. Since the cell fragments floated in a volume of fluid large compared to their size, and since a rehtively low density of pigment exists in a single outer segment, we can safeIy conclude that the data reported are not of thermal Origin.
Two types of measurements were taken on each photograph selected for analysis: (1) The fractional fringe shift at the center of the outer segment. This gives the difference in optical path length through the center of the outer segment compared to that of an equal thickness of reference path through the medium. (2) The width of the outer segment was measured between corresponding points at the edges of the cell where the rate of change of fringe shift was a maximum. Taking the latter measurement to represent thickness as weI1, the two measurements together give the difference in average index of refraction between the interior of the rod and the medium. The immediate objective of the experiment was to compare the resulti of such measurements before and after bleach. Unfortunately, interference microscopy poses certain measurement problems. Best interference is achieved by stopping down the aperture of the condenser lens, but this reduces resolution (ALLEN, B~MJLT and ZEH, 1966). The former is needed for evaluation of fringe displacement and the latter for dimensional measurements. Actual experimental arrangements necessarily represent a compromise. Data reduction
A large number of specimens WBSstudied to obtain our results. It WBSfound necessary to reject most sets of photographs. Any one of the following criteria was sufficient for rejection: (1) When the time between decapitation and completion of data collection exceeded 6 min. The intent was to minimize postmortem effects (e.g. ENOCH, 1966, 1967b).
1-4
J.~Y XI. EXOCH, JOHN SCANDREIT .AND FRANK L.
TOBEY.
JR.
(2) When the cell moved (lateral or rotary displacement) during determinations, or was not centered in the field (some pincushion distortion was present near the edge of the field), or was detectably not set perpendicular to the fringe pattern. (3) When the cell exhibited noticeable imperfections, or did not have an attached ellipsoid. (4) When exposures were incorrect or not uniform throughout the series, or development resulted in excessive graininess. (5) When focus was incorrect, or resolution, or fringe definition was faulty. (6) When immersion medium index of refraction determinations departed from mean determinations by a significant amount. No photographs were rejected on the basis of computed results. The optical path difference was computed from the evaluated fringe shift (Fig. 1). B = ; A, (A = 0.826 !.L). The thickness (d) in microns, assumed to be equal to the width, was derived by comparison of the measured d with a stage micrometer photograph also taken at 826 nm. Then the index of the outer segment (n,) is given by 6 = [nz - n,]d
8
n, = - _t n,
d
where n, is the index of the immersion medium (Table 1). A computer controlled microdensitometer was used to facilitate the analysis. The instrument consists of an optical image scanner connected to an IBM 360-50 computer. A cathode ray tube serves as a randomprogrammable point source light addressed by the computer on a 8192 x 8192 point grid. The imaged radiant energy passing through each 35 mm data film point is collected at one photomultiplier and the transmitted signal is compared with that of a reference photomultiplier measuring the output of the source alone. The logarithm of the ratio (transmittance) was digitized on a O-63 numerical scale and returned to the computer. The basic point measurement cycle required 20 @sec. A magnified display of a 256 x 256 grid of points above a hxed numerical threshold is shown in Fig. 2. The rod outer segment was seen by the densitometer only as a systematic disturbance in the interference fringe pattern. The fringe separation was measured by scanning across two fringes immediately to the side of the outer segment with a line scan parallel to the cell wall. A linear density array of 200 points was produced and the centers of the two density peaks were computed by forming first moments of the peak shapes. The fringe displacement was determined by scanning across a displaced fringe in the center of the outer segment and scanning in a parallel direction across the undisturbed fringe on each side. The fringe peaks were located by forming hrst moments of the 200 measurements used in each scan. The fringe shift was obtained as the displacement of the center peak in a direction perpendicular to the line joining the two side peaks. The fractional fringe shift of each frame was taken as the mean of the individual quotientsn/b as obtained from ten repeated measurements of each pair taken on 10 separate days. Typical values were about 0.5 fringe and the mean precision of all determinations was 0.33 per cent. Individual precisions are included in Table 2. Measurements of outer segment image width were obtained with an elaborate scheme for producing a maximum-line-of-disturbance signal. The cell edge was detined as that line which had a maximum average transverse density gradient, where positive and negative gradients were separately averaged, then maximized. The length and initial location of a trial edge line were imposed by the operator who controlled the locations of cursor points on a computer-displayed image of the cell fringe pattern (Fig. 2). At each of 50 equally spaced points along this line, a transverse density scan of 11 points was performed, and a least-square fit to the transverse density was used to estimate the density gradient. The 50 transverse density gradient values were combined into separated positive and negative gradient signals. In Fig. 2, one notes that along the lateral border of the outer segment, dispIaced fringes result in alternate positive and negative transverse gradientshence the utility in separating the two signs of gradient signals. As the operator varied the initial lateral displacement and angular orientation of the trial line, the computer repetitively performed the scans, fitted, averaged and displaced the numerical values. The operator oriented the line to approximately maximize the sum of the positive and negative signals. The computer used this line as the starting point in an automated search procedure. The line was stepped in 1” rotations through 5 5” about the initial line. The 11 values of average density form peaks whose centroids were located separately for the positive and negative signals. The line was then reoriented to the optimal angle, and 11 transverse steps of lateral position were made over a range approximately equal to half an outer segment width. The gradient signal values again form peaks whose centroids determine the final ‘best-fit” line location and orientation. This process was repeated for the other lateral border of the outer segment. The final result
FIG. 1. Schematic drawing of a frog rod preparation as viewed through the microscope. Fringe separation, b, and fringe displacement, a, are measured. From these data and the test wavelength (826 run), difference in optical path length, 6, can be computed. Assuming width change is equivalent to diameter change in a cyiindrical rod, path length ditference through the cell and index of refraction of the immersion medium allows a determination of outer segment index of refraction (x2) along the test beam. E-vector was perpendicular to the long axis of the rod in these experiments.
FIG. 2. Polaroid photograph of microdensitometer display during analysis. [facing page 174
B!esching
E?fect on Frog
I75
Otctzr Segment
TABLE 2. DETERVIUATIOM A\D CALCULATIOLS
Specimen
Time
~__ Ffame No. I
Average Average
55B 58D 58E 58H 59B 59A 72E
04018 o-4452 04487 04419 0.4746
_ Prebleach ____0.41 0.23 0.29 0.18 0.25
5’13” 3’43” 2’50” 3’32” 5’31” 5’55” 1’32”
of 5 of 7 5’16” 3’46” 2’54” 3’35” 5’34” 5’58” 4’35”
of 5 of 7
0~4000 04410
0.36 0.15 0.34 0.31 0.18
0.4468
0.4624 0.4830
0.4139 04458 0.4504 04476 0.4728
0.53 0.27 0.19 0.29 0.19
04461
.^ _ Frame No. 4
Average Average
55B 58D 58E 58H 59B 59A 72E of 5 of 7
Frame No. 5
Average Average
5’41” 4’06” 3’17” 3’55” 5’52” 6’20” 4’51”
of 5 of 7 5’44” 4’12” 3’21” 3’58” 5’55” 6’23” 4’54”
1.33061 I.33061 1.33061 1.33000
1.-LO66 1.3934 1.396-! 1.39isl 14052
1.3995
5.21 7.08 6.78 6.70 6.34 5.72 5.10 6.12 6.13
0.06 0.12 0.12 0.27 0.20
5.31 7.05 6.72 6.65 6.28 5.74 5.14 6.38 6.11
0.09 0.13 0.22 0.33 0.19
04053 0.4336 0.4578 04419 O-4782
04038 04469 0.458 1 04440 0.4761
04457
1406’ 1.3928 1.3979 1.3996 1~40610
1.4005
1.32951 1.33061 1.33061 1.33061 1.33000
1.4OS9 1.3938 I .3976 1.3979 14052
1.4006
-Y-77---
Postbleach 04073 04400 0.4535 04424 0.4734
1.32951 1.33061 1.33061 1.33061 1.33000
second bleach 069 0.20 0.22 0.97 0.20
0.60 0.20 0.26 0.37 0.30
04433
55B 58D 58E 58H 59B 59A 72E of 5 of 7
10
04433
55B 58D 58E 58H 59B 59A 72E
Frame No. 6
Average Average
__~____ 5’38” 4’03” 3’14” 3’52” 5’49” 6’17’ 4’48”
0.14 0.15 0.15 0.19
6.13 6.14
0.4486
55B 58D 58E 58H 59B 59A 72E
5.21 7.08 6.81 6.74 6.31 5.12 5.i 1
04424
558 58D 58E 58H 59B 59A 72E
Frame No. 3
Average Average
5’10” 3”O” 7 2’44” 3’29” 5’28” 5’52” 4’29”
of 5 of 7
Frame No. 2
Average Average
-.___
0.35 0.34 0.31 0.35 0.24
5.35 7.16 6.85 6.72 6.37 5.71 5.20 6.49 6.20
0.16 0.11 0.20 0.20 0.18
5.29 7.09 6-83 6.73 6.37 5.71 5.13 646 6.16
0.19 0.14 0.26 0.08 0.12
5.31 7,04 6.85 664 6.37 5.76 5.15 6-44 6-16
0.35 0.21 0.23 0.18 0.08
-______ 1.32951 1.33061 1.33061 I.33061 1.33000
14056 1.3920 1.3968 1.3964 14043
I.3990
1.32951 1.33061 1.33061 1.33061 1.33000
14061 1.3917 1.3976 1.3962 14050
1.3993
1.32951 1.33061 1.33061 1.33061 1.33000
14055 1.3940 1.3974 1.3974 14047
1.3998
176
JAY M. ESOCH, JOHN SCANDRETT AND
5 4
;
FRANC L.
TOBEY, JR.
19sec
;jmln
IO
30 Time,
53
set
FIG. 3. Means of ten microdensitometer readouts of cell width taken on the three photographic frames recorded just before and the three just after a lo-set white light bleach. d is plotted in microns. The arrow gives the time from decapitation to the last photograph before bleach. displayed to the operator was the outer segment taper in degrees and diameter, defined as the spacing between beat-fit centers. For a typical outer segment 10 replications of the width measurement, taken on different days, showed a 0 of O-18 per cent. In order to combine data from several samples, an adjusted time scale had to be established. Each photo-
Time,
set
FIG. 4. Combined means of d for all seven ceils from Fig. 3. It is apparent that exposure to light caused an increase in outer segment width.
177
Weaching Effect on Frog Outer Segment
graph from each preparation was taken at a different time after decapitation, and each bleach was initiated at d&rent moment on each sample. Times for the individual frames were averaged, and these mean times were plotted relative to the average time of onset of the bleacfimg light (e.g. Fig. 4). WhiIe twenty exposures were taken on each sample, only the six taken immediatelybefore and after the bltich were actually anaiyzed instrumentaily. It was found that this procedure reduced judgment errors by the operator who was actively involved in locating reference measurement points on the storage CRT display. Many hours of computer time were required for the analysis of each cell. RESULTS Ofthe many receptors studied, onfy seven met the stringent requirements outtined above for measurement of outer segment width. Two of these had minor imperfections which interfered with determinations of optical path length. Table 2 gives the mean data on each exposure of each sample and the derived quantities. Figure 3 shows the width measurements made on the seven acceptable sets of photographs, and Fig. 4 shows the mean width data. AIthough the observed changes are superimposed upon on going changes due to deterioration of the dying cell fragments (e.g. ENOCH,1967b), it is nevertheless clear that a small increase in width occurred following the bleach. u/dfor most width determinations was about O-2per cent (Table 2). Thus representative vaIues of sigma were about 0.012 p or 120 A, little more than the thickness of a unit membrane. Conside~ng the fact that the measurements were made at 0,826~ and that the numerical aperture of the interference microscope was reduced below optimum, this is a precision well beyond that usually expected in microscopy.
3 mm 46sec 4 3 0 3
x
-0 -
0,440
o
d
*3mfn f
0
.C 8 g * I I!
54sec 0.460 -Zmin o 4
58D
0
_
0.450
0 - 0.430 58E ooo i ui
34~
58H 0.460
0 0
0,490
-
00
“I o
0
- 0440
Smin 34sec
04?0-
a
0 00
(I I to
598
i 20 Adjusted
t 30 time.
I 40
I 50
1
set
Difference in optical path length (8) before and after bleach. Mean data from ten independent readouts from each of three frames recorded just before and three just after the IO-see white light bleach. Five of the seven cells from Fig. 3 were used. FIG. 5.
V.I. 1311-M
i78
JAY M.
ENOCH,JOHN
SCAXDRE~
ASD FRANK L. TOBEY, JR.
In those figures referring to individual cells, a common time scale has been plotted, with the actual time of one photograph indicated. The time after decapitation is included in Table 2. Figure 5 shows the optical path length difference of the acceptable specimens. On the basis of these individual records and of the mean data (Fig. 6), it is evident that little or no change occurred in optical path length difference following bleach.
Mean
measuredfactors
Scells 82Snm
0
I
0
.f=Z?C
IO 23 30 Adjusted rrme, set
,, IO Adjusted
40
i2,i 20
ttme,
3( see
FIG. 6. Combined summary data for five cells. Outer segment diameter and optical path length
axe measured quantities. Outer segment index of refraction (n2) is derived.
Two approaches may be used in evaluating Fig. 6, neither fully satisfactory. The mean of the three values before bleach may be compared with the mean after bleach. This tends to overlook differing secular behaviour in the cell fragments, and treats all variation between individual frames as due to measurement unce~ainty which is not correct. There is remarkably small variance in our analysis techniques. A second alternative is to project a trend from the prebleach to beyond the bleach period. We feel there is too much uncertainty in any apparent trend to warrant this approach. Nevertheless, from simple inspection of Figs. 4 and 6, one can estimate that on an average, bleaching induced an increase in diameter of 2-4 per cent. Sirnilariy, we conclude that there is probably less than a I per cent change in optical path length. Thus the average index of the outer segment must have decreased on the order of 0*1-@2 per cent following a bleach. (2-4 per cent change in index difference.> Taking the measured width to equal the thickness, we obtain a value of about 1400 for she absolute index of refraction of the frog outer segments. This compares reasonably well
Bleaching Effect on Frog Outer Segment
179
with the value of l-411 previously reported by SID.MAN(1957), particularly when one considers the test wavelength difference and the fact that Sidman did not consider postmortem changes or light induced effects. DISCUSSION
Our results indicate that the changes observed here should have very little effect on the waveguide properties of the frog outer segment. For example, the numerical aperture of the fiber, given by ff3 sin a = t/&’ - nlz) is virtually unaltered by bleach. In this equation, n3 is the index of the medium preceeding the fiber optics element, and a is the largest angle of incident light containedby total reflection within the fiber (e.g. ENOCH, 1963). The fact that a limiting numerical aperture exists for fiber optics elements (such as retinal receptors) indicates that a11are directionaiiy sensitive to some degree. An important factor which, in part, describes the transmission capability of a cylindrical waveguide is its cutoff parameter (SNITZEXand OSTERBERG, 1961) p = ;
y’(n*2 - It,*),
where d is the diameter of the waveguide, A is wavelength in vacuum, and nZ and n, are the refractive indexes of the core (outer segment contents) and cladding (interstitial matrix or medium) respectively. The cutoff paramater is simply the product of the waveguide circumference in wavelength units times the numericaf aperture of the guide. Each waveguide modal pattern itself is characterized by a cutoff parameter u,,, which is a particular value of the arg~ent of the Bessel function J_(u) describing the radial dependence of the E(electric) and H(magnetic) vectors of the mode. u,, is that value for which J,(ur/O-56) goes to zero_ Here r is the radial coordinate, and u is a parameter which fixes the scale of the Bessel function relative to the diameter of the core, d. A waveguide may transmit any mode for which
However, this relation does not determine which mode (or modes) is actuafly transmitted in a given case. ENOCH(1963) gives a list of low order modal parameters. Our data indicate that the observed change in d would tend to increasep by 1-3 per cent while the change in n, would tend to decreasep by O-S-2 per cent. Using mean values (5 cells) from Table 2, the resultant value of p increases by O-45%. Changes inp alter the wavelength of cutoff induced transition from one mode to another. A change in modal pattern alters the energy distribution and hence the absorption to some extent. This could lead to local perturbations of the absorption curve which would effect the difference between pre- and post-bleach spectra. Certainly, however, the effects should be minor. We can compute from the above expression for p that changes of the observed magnitude would shift the transition wavelengths on the order of 2-3 nm, which is near the resolution limit of most ~~os~trophotomet~ of visual receptor cells. The difference in optical path length between the frog outer segment and the aqueous surround is primarily due to the high molecular weight protein, phospholipid and lipid constituents present in the highly organized membrane and opsin rich outer segment. On the other hand, the concentration of high molecular weight constituents is low in the aqueous
180
JAY M. ENCH, JOHNSCANDRETT AND FRANKL. TOBEY, JR.
humor, as may be inferred from the index of refraction of the immersion medium which hes close to that of water (Table 1). &RER (1957) derives concentration of solids in individual parts of retinal receptors, using the relationship: C - n2 - no, a
where C is total concentration of solids in mass per unit volume (g/l00 ml), and no is the index of the solvent. The latter may be interchanged with nt, if n, approaches n, (R. BARER, 1957, 1966). a, the specific refraction increment, is treated as a constant for a given ty-pe of biofogical material and has units of volume per unit mass. If n, = n,, 6 = (n2 - n,)d = aCd.
Strictly speaking, this relationship applies to homogeneous cells of uniform thickness (BARER,1966). As indicated below, one must use care in its appii~tion. It is worth consideling briedy the nature of the changes taking place. Let the prebfeach condition be designated by the subscript x and postbleach by y. Our findings can be summarized by 6X= a,, and From above
d y = 1.03d,
aCJX = aGydp,
assuming that width = thickness. It follows that C& = C,( 143dJ
c,
=
l.O3C,
This leads to the conclusion that average concentration of solids, C,, decreased by about 3 per cent. Assuming low index material moves iuto the outer segment, cross sectional area and volume per unit length should increase by 6 per cent, and hence average concentration would decrease by the same amount, neglecting changes in length. Change in length can not be neglected, of course, in estimating total change in volume. The observations could be explained if a 3 per cent increase (say) in diameter were accompanied by a 3 per cent decrease in length. Net volume increase would be 3 per cent resulting in a 3 per cent decrease in refractive index difference or a change just sufEcient to cancel the increased geometrical path. ~~o~~te~y, length was not measured in this study as we were interested primarily in waveguide effects, and a waveguide comparable in length to the outer segment cau be regarded as Snitely long. In any case, the method employed is not suitable for precise length measurements. WEALE(197Ob, 1971~) has suggested that bleaching causes a slight shortening of the outer segment. His estimate ranges between O-02and O-05 pFor a frog outer segment 40 p long, a reduction by 0.05 p would reduce the total volume by only O-1 per cent which is negligible compared to the effects reported here. We have considered another simplified model consistent with the observed data. If bleaching causes water to enter through the ceil membrane, r, but the water remains as a hollow cylinder about the disc core, diameter would increase, but measured difference in
Bleaching Effect on Frog Outer Segment
181
optical path length would not change, since the concentration and thickness of high molecufar weight constituents would not be altered. In this model waveguide properties would not change. We have implicitfy assumed above that the narrow ciiium acts as at least a partial barrier to inflow from the medium via the fractured inner segment. Moreover, in a recent study, CO~IE?J(1971, see his Fig. 2) shows that isolated outer segments from retinas exposed to hypotoic solutions retain their swollen form, suggesting that the cell membrane broken in the process of separation of the fragment acts as if it were healed. ~uwt~nm (I 970) has obtained scanning electron micrographs of rat and monkey retinas follofing intense, long duration exposures to light (indirect ophthalmoscope, for up to 4.5 min). His prints appear to show much greater swelling of outer segments than we have observed, as well as irregularities in the surface and vacuole formation in the Iamellar discs. If these gross dimensional changes are real, one could predict that retinal detachment must accompany such extreme exposures unless there is compensatory shrinkage of the interstitial matrix. The irregularities should result in markedly increased light scatter (MARCUSE, 1969). We have observed nothing resembling these changes in our- experiments. It is clear that more sophisticated models are required to explain the effects of bleaching on receptor outer segments. In particular, the models presented above do not account for all changes in birefringence (WEALE, 1971a,b,c,d) conformational changes (HELLER, 1968) or swefling behavior (COHEN, 1971). Neverthetess, it appears that the effects observed here should not seriously interfere with attempts to characterize the transmission properties of the receptor or the absorption spectra of photolabile pigment in single receptors by microspectrophotometry. .&kno&dgements-Dr. HARRYQUI~LEYand Miss EVA BACHMANN assembled the test data. Mrs. JOAN CRAMER O’HAIRand Mrs. BSWZLYLAWWZNCE performed the final computations on the IBM 360 coupled m~roden~itomcter under Dr. SCAXDRETT’S guidance. Dr. HORN PALERof Zeiss assisted in solving some of the compIex optical problems encountered. The authors wish to thank Dn. R. BARER,B. BECKER, S. E~NTWG,A. COHEN and N. DAW for critically reading this manuscript. REFERENCES ALLEN,R., BRWLT, J. and ZEH, R. (1966).In Advances in Dpticai and Electron Microscopy (&ted by R. BARER and V. Coss~m~), Vol. 1, pp. 81-87.Academic Press. London. BARER,R. (1957). Refractometry and interferometiy of living ce&. J. opt. Sac. Am. 47,545-556, BARER.R. (1966). In Physical Techniques in Biologicd Research (edited by A. POLLI~R), Vol. 3A, pp. 44. Academic Press. New York. COHEN,A. I. (1971). Electron microscopic observations on form changes in photoreceptor outer segments and their saccules in response to osmotic stress. J. ceil &oL 48, 547-565. ENOCH,J. M. (1961).Nature of the transmissionof energy in the retinal receptors. J.opt. sot. A~. 51, 1122-I126.
ENOCH,J. M. 0963). OpticaI properties of retinal receptors. .F. opt. Sot. Am. 53,71-85. ENOCH,3. M. (1%7a). The retina as a fiber optics bundle, Appendix B. In fiber Optics, Pr[nc@/es d Applications (edited by N. S. KAPANY), pp. 372-396. Academic Press, New York. (An updated edition should appear in 1972). ENOCH,J. M. (1967b). Comments on “excitation of waveguide modes in retinal receptors”.J.opt. sot.Am_ 57, 548-549. ENOCH,J. M. and GLSMAW L. (1966). Physical and optical changes in excised retind tissue. Imestne, Oph~~~. 5,208-211.
FELDMAN,E. ad KWUWRA, T. (1968).The retinal pigment epithelium. XV.The damaging effects ofradiant energy. A.M.A. Archs Ophrhal. 80,265-279. mRO% F. (1971). Frog Rhodopsrir in situ: OrientatioMIandSpectraral Changes in the Chromphores oflsofared Rerimf Rod ceik Dissertation. The Johns Hopkins University, Baltimore, Md. HFJLr-WJ. (1968).Structure of visualPigments II. Binding of retinal and conformational changes on light exposurein bovinevisual pigment(Xmax = 500 nm). Biocfremistry 7,2914-292o.
JAY Xl.
182
ENOCH,JOHNSCANDREI-~ AND FRAXKL. TOBEY,JR.
J. (1966). Some experiments with a photoelectric ophthalmoscope. In Performance of the Eye or Low Lumenunces (edited by M. BAUMANand J. Vos) pp. 171-181. Exerpta Medica Foundation,
KRMJSKOPF,
Amsterdam. Km, W. (1877). Veranderungen der Stabchen durch Licht. Untersuch, a.d. Physiologischen Inst. d. Univ. Heidelberg, Band 1, Heft 1,409-411 (Cites the work of F. VONHo-). KS, T. (1970). Retinal recovery from exposure to light. Am. J. Ophthaf. 70, 187-198. LATES, A. and L~EBEMAN. P. (1970). Cones of living amphibian eye: selective staining. Science, N. Y. 168, 1475-1476. MARCUS~, D. (1969). Mode conversion caused by surface imperfections of a dielectric slab waveguide. Bell
System Tech. J. 48,3187-321X OHZU, H. and ENOCH,J. M. (1972). Optical modulation by the isolated human fovea. Vision Res. 12,
245-25 1. OIUU, H.. ENOCH,J. and Q’HAIR,J. (1972). Optical modulation by the isolated retina and retinal receptors. vision Res. 12,231-244. POLE,R. et al. (1972). Topical Meeting on Integrated Optics-Guided Waves, Materials and Devices. Las Vegas, Nev. Feb. 7-10, 1972. Optical Society of America, Washington, D.C. &MAN, R. (1957). The structure and concentration of solids in photoreceptor ceils studied by refractometry and interference microscopy. J. biophys. biochem. Cytol. 3, 15-20. Sm, E. and OSrEmERG, H. (1961). Optical dielectric waveguide modes in the visible spectrum. J. opt. Sot. Am. 51,499-505. SNYDER,A. and RICHMO~-D, P. (1972). Light absorption in visual photoreceptors. Science, N. Y. (submitted for publication). WASHBURN, E. (editor) 1930. ZntemationaICritical Tables of Numerical Data Physics, Chemistry and Technology. (1st edition), Vol. 7, p. 13. McGraw-Hill, New York. Wute, R. (1964). Comparisons of reactions of human and rabbit fundi to photopic exposures. J. opt. Sot. Am. 54,120-126.
WEALE,R. A. (1965). High light intensities and photo-chemical reactions of human visual pigments in situ. In Symposiu on Quunt. Biol. Vol. 30, pp. 335-343. Cold Spring Harbor Symp. Quant. Biol., New York. WHALE,R. A. (197Oa).Optical properties of photoreceptors. Br. med. Bull. 26,134-136, plate 9. WEALE,R. A. (197Ob).How the birefringence of vertebrate rods is affected by light. J. Physiol., Lond. 210, 28-29P. W~ALJ$R. A. (1971a). On the linear dichroism of frog rods. Vision Res. 11,1373-1385 (Appendix by R. W* SMlTH). WEALE,R. A. (1971b). Rod birefringence and light. VisionRes. 11, 1387-1393. WEALE,R. A. (1971~). On the birefringence of rods and cones. Pmers Arch. ges. Physiol. 329,244-257. Wg,ua, R. A. (1971d). Photolysis and birefringence of frog rods. Experientia 27,403. WOLKEN, J. (1963). Structure and molecular organization of retinal photoreceptors. J. opt. Sot. Am. 53, I-19.
Ah&act-An i&a-red interference microscope has been developed to study the effects of a white light bleach on physical properties of single receptors. The width and optical path length was measured for tie& ftog rod outer segments lying on their sides before and just after a major bleach. The data presented were recorded some seconds after the bleach. Use of a computer controlled microdensitometer allowed determination of these parameters with precision well beyond normaI resolution limits. Following light exposure, receptor width increased between 2-4 per cent and optical path length difference remained virtually unchanged. This suggests that a small decrease in index of refraction occmred in the receptor outer segmentat the wavelength used (826 nm).
R&umGQn a construit up microscope interferentiel infrarouge pour I’etude des effets de la lumi&e blanche sur la prop&& physiques de.s r&epteurs isoiis. On mesure la largeur et le trajet optique de segments extemesfraisdeb&onnetsde grenouilles,couch&ur leursc&&,avant et juste apr&s une d&oloration intense. Lea don&es etaient enregistrees quelques secondes aprts Ia d6coIoration. Un microdensidom&re contr~le par une cabdatrice permetde d&miner ces parametres avec une pr&ision largement sup&ieure aux limitee normales de r&solution. Apt& exposition a la lumiere, la largeur du recepteur augmente de 2 a 4 pour cent et le trajet optique reste pratiquement constant. Cela suggere une petite diminution de l’indice da refraction dans le segment exteme du r&epteur pour la longueur d’onde employ&z (826 nm).
Bleaching Effect on Frog Outer Segment
ZusaMnentaswng-Urn
! 83
die Effekte zu untenuchen, wenn man mit weissem Licht die natiir-
lichen Bestandteile von einzelnen Rezeptoren bleicht, wurde ein Infrarot-Interferenzmikroskop entwickelt. Gemessen wurden die Breite und die opt&he Wegllnge von aug der Seite liegenden Susseren Froschstibchensegmenten vor und nach einer grijsseren Bleichung. Die sich dabei ergebenden Messwerte wurden einige Sekunden nach der Bleichung aufgezeichnet. Durch
Verwendung eines rechnergesteuerten Mikrodensitometers konnten diese Parameter mit einer Genauigkeit weit unter der Gblichen AtiBsung bestimmt werden. Nach der Lichteinstrahlung verbreiterte sich der Rezeptor um Z-4 Prozent, wshrend die opt&he WeglrIngendifferenz unvedndert blieb. Das Itit vennuten, dass die Brechzahl im Busseren Rezeptorsequent bei der verwendeten Wellenllnge von 826 nm etwas kleiner wird.
Pe3lOMe-&Il pa3pa60TaH kit$paKpaCHblk HHTep@e~HUHOHHbltiMHKpOCKOU, ttSUIH3y’IeHUR neficrerur 06ecuaeraeamfn 6enbrM C~OM Ha &isarecKae csoBcrea enHAEiyHb IX peUeIlTOpOB. 6buni w3hfepem msipmia x nmisra orrr~~ec~oro nyni nnx caeamx Hapyxaibtx CerMeirroa
nanoser
n5uyrma3 nepea
H cpa3y rfocne Bommoro
06ecuaexsxaa~~n.
llpenixaanemib~e
naHxbte perncrpaoBaawcb sepes HecKonbKo ceKyHn nocne 06eco~e~~~a~xx. klcnonb30aaH~e KobfubioTepa Kom-pomipymqero MmcpojxeHcxToMeTp n036oNUIo 0npenenm-b ~TH rrapaMeTpbl C TOYHOCTbIO 6n~31rotiK HOpMaItbIibtMrpaHZiUaMpa3~meIiEX. llocne 3KCKIO3ELtIiH K CeeTy nmpmia peuerrropoe yaenn-93mTacb B npenenax 2-4x, a pa3nmRn B Anmie OrrrwecKoro ny~11:aa_ecKa ocTaeamicb ~eB3MetIRbIMB. 3~0 AaeT OcHORaaJieQfETaTb, ‘IT0 He6onbmoe yMeHbmeHwe nOKa3aTenl npenowrefim 803HmcaeT B HapmOM CeMeHTe ~UetITOpa IlpH HClTOJIb30BaHHO~ LLmiHeBOLlHbI(826 AM).
INTRODUCTION RETINALreceptor cells behave like dielectric waveguides for electromagnetic radiation at optical wavelengths (e.g. ENOCH, 1967a, KRAUSKOPF,1966). A waveguide operating at wavelengths in a range comparable to its diameter does not transmit all WaveIengths unifo~ly, Hence an effective spectral absorption curve taken end-on though a receptor may
differ in detail from the absorption of isolated pigment, even allowing for pigment dichroism (e.g. ENOCH,1961). At a wavelength near a cutoff transition of a modal pattern, a cylindrical waveguide is critically sensitive to diameter and to the diference between the indexes of refraction of the core and the exterior medium, especialiy if the difference is smalf, as is usually the case for biological material. It becomes important to know if changes induced in the receptors, either in vitro or in vim, can produce changes in their waveguide behaviour. For example, this can be a significant question in through-the-retina microspectrophotometric studies. In the work described here, we have measured the widths of rod outer segments and their index of refraction along a path perpendicular to the long axis before and after bleaching. Isolated frog rods (Rana pipiens) with attached ellipsoid (fractured in the inner segment) were examined with a Zeiss interference microscope modified to function in the infra-red. All measurements were made at h = 826 nm in the infra-red in order to prevent bleaching by the measuring beam. F. von Hornbostel, (cited by KUHNE, 1877), first suggested that an increase in frog receptor diameter might be caused by the bleaching of the photolabile pigments. WOLKEN (1963) also reported a similar result. More recentIy FRIEDMANand KUWUBARA(1968) and KUWUBARA(1970, and personal co~unication) provided electron microscopic evidence of swelling and other physical changes induced by the action of light occurring in photoreceptors of rats and monkeys. WEALE(1970a, b) has studied the birefringence of frog retinal receptors and deduced from his results that changes inthe length of the receptor occur following bleaching. More recentiy WEALE(197Ia, b, c, d) has obtained data on frog rod birefringence and has provided an analysis of the components contributing to birefringence in the dark adapted and bleached states. He observed differences in index of refraction in the I This work has been supported in part by Research Grant No. Ey 00204 and by Career Development Award No. K3 15138 (to J.M.E.) of the National Eye Institute, and by Research Grant NO. RR 0396 of the Division of Research Resources (to -IS.), National Institutes of Health, Bethesda, Maryland. 171
111
JAY Xl.
ENOCH, Jon
SCAXDRETT AND FRASK L. TOBEY, JR.
third and fourth decimal place. SNYDER and RICHAIOND (1972) present a theoretical treatment of the effect of anomalous dispersion on receptor index of refraction and photolabile pigment absorption. Many authors in recent years have dealt with physical and chemical changes occurring in the outer segment due to the action of light. METHOD Apparatus A Zeiss interference microscope (Standard Universal [POL] Jamin-Lebedeff type) was adapted for use in the infra-red. A 150-W Osram Xenon arc was substituted for the standard Zeiss Mercury arc. A RairdAtomic infra-red interference filter (passband of 820-832 nm) was used at the field stop to provide a monochromatic beam. A calcite polarizer in a three-point mount replaced the subcondenser Polaroid in order to improve polarization and transmittance in the infra-red. Since the polarizer and interference filter were removed during bleaching, the three point mounting allowed rapid relocation in the dark. The quarter wave plate was replaced by one designed for X = 826 nm. An analyzer eyepiece (Zeiss No. G41-452 with special Wollaston prism elements) was used to introduce fringes in the field (Fig. 1). The 40x condenser and objective units were retained. The resultant field was imaged by a photoeyepiece onto the photocathode of an RCA 6914-A infra-red image converter fitted with an Et-Se eyepiece to magnify the image on the phosphor screen. An Exacta camera was used to record the phosphor display. The time of each photograph and of bleach duration were recorded on a constant speed penwriter. A Leitz stage micrometer provided dimensional calibration. The E-vector of the measuring light was set perpendicular to the long axis of the outer segment and parallel to the fringe pattern. Thus the E-vector lay in the plane containing the absorption axes of the visual pigment, but not in the absorption plane of certain photoproducts (e.g. HAROSI,1971). Sample preparation
Since it was expected that only small changes in size and optical path-length might result from bleaching, frog rods were used because of iheir size. Considering the resolving power of the light microscope at X = 826 nm, our ability to detect small changes in a mammalian receptor having a diameter of 1.5-2.0 p appeared questionable. Frogs were dark-adapted overnight. Time recording began with decapitation of the frog, after which a single razor slice served to remove the anterior half of the eye. The retina was removed with iris forceps and lightly dipped in the immersion medium to detach a small number of receptor elements. Only outer segments with attached ellipsoids were used, and measurements were limited to the outer segment. All dissection was conducted in total darkness with the aid of a second infra-red image converter and infra-red sources. Every effort was made to diminish the elapsed time between dissection and measurement in order to minimize artifacts in these preparations. Initially, aqueous humor from the second eye of the frog was used as the immersion medium for the rod preparation. Care was used to prevent evaporation of samples by storing them in closed chambers slightly larger than the drop volume. When it proved ditTicult to obtain sufficient aqueous from the second eye, fresh aqueous humor from the eye of a large goldfish was substituted without apparently altering the results significantly. Since tricaine solution was used to anesthetize the goldfish, it was necessary to carefully blot the eye in order to prevent contamination. The index of refraction of the immersion medium was measured with a temperature controlled Bausch and Lomb Precision Refractometer held at the average room temperature of 22’C. Our measurements of triple distilled water taken under standard conditions (589.3 nm, 20°C) matched standard values well. Significant values are listed in Table 1. All index values measured at h = 589.3 nm were corrected to the test wavelength of 826 nm (WASHBURSE, 1930). This correction assumes that the dispersion of the index for aqueous follows that of water. This is reasonable, since the index at 589.3 nm is close to that of water, and both fluids are transparent over the relevant wavelength range. Experimerltal procedure
Ten exposures of the interference pattern were taken in rapid sequence, followed by a IO-see bleach. Ten more exposures were taken immediately after the bleach. The aperture of the condenser was not altered during the bleach. Since the white bleaching light was observed to grossly bleach a fresh retina placed on the stage, and since thcrmopile (Eppley) measurements indicated that the energy/unit area delivered to the specimen should have been adequate to bleach all rhodopsin, bleaching was assumed to be complete. Sample heating is not a serious problem with this instrument since standard microscope optics filter out radiation of h > cu. 900 nm. As a check, temperature rise during a bleach was measured with a Yellow Springs recording microprobe thermistor placed in the light path on the microscope stage. Less than 1°C
173
Bleaching Efkct on Frog Outer Segment
TABLE 1. 1. Mean
INDEXOF REFRACTION
of values measured at 589.3 nm Aqueous humor
Temperature (“C)
Pure Hz0
Frog
Goldfish
Tricaine solution
20 21 22 23 24
l-33307 i-33301 1.33290 1-33282 1.33277
1.33609 l-33601 1‘335% 1.33587 1.33582
I.33499 l-33497 1.33486 1.33477 1.33466
1.33323 I.33318 l-33307 1.33296
---_ 2. Refractive index, no, of pure water at A = 826 nm source: International Critical Tables (1930) x n0 589.3 826
I.33300 1.32780
t
20°C 20°C
3. Index of refraction of measured specimensL xl adjusted n, measured (t = 22°C; II = 589.3 nm) (r = 22°C; X = 826 nm) Test day _. __--- ____ .._ 55 1.33472 1.32951 ii
1.33522 l-33527
1*33ooo 1.33061
72
1.33467
1.32945
r Values for fresh gold&h aqueous humor sample used with given frog retinal specimens on specific test days.
change in temperature was recorded in the plane of the specimen during a IO-se-cexposure. Since the cell fragments floated in a volume of fluid large compared to their size, and since a rehtively low density of pigment exists in a single outer segment, we can safeIy conclude that the data reported are not of thermal Origin.
Two types of measurements were taken on each photograph selected for analysis: (1) The fractional fringe shift at the center of the outer segment. This gives the difference in optical path length through the center of the outer segment compared to that of an equal thickness of reference path through the medium. (2) The width of the outer segment was measured between corresponding points at the edges of the cell where the rate of change of fringe shift was a maximum. Taking the latter measurement to represent thickness as weI1, the two measurements together give the difference in average index of refraction between the interior of the rod and the medium. The immediate objective of the experiment was to compare the resulti of such measurements before and after bleach. Unfortunately, interference microscopy poses certain measurement problems. Best interference is achieved by stopping down the aperture of the condenser lens, but this reduces resolution (ALLEN, B~MJLT and ZEH, 1966). The former is needed for evaluation of fringe displacement and the latter for dimensional measurements. Actual experimental arrangements necessarily represent a compromise. Data reduction
A large number of specimens WBSstudied to obtain our results. It WBSfound necessary to reject most sets of photographs. Any one of the following criteria was sufficient for rejection: (1) When the time between decapitation and completion of data collection exceeded 6 min. The intent was to minimize postmortem effects (e.g. ENOCH, 1966, 1967b).
1-4
J.~Y XI. EXOCH, JOHN SCANDREIT .AND FRANK L.
TOBEY.
JR.
(2) When the cell moved (lateral or rotary displacement) during determinations, or was not centered in the field (some pincushion distortion was present near the edge of the field), or was detectably not set perpendicular to the fringe pattern. (3) When the cell exhibited noticeable imperfections, or did not have an attached ellipsoid. (4) When exposures were incorrect or not uniform throughout the series, or development resulted in excessive graininess. (5) When focus was incorrect, or resolution, or fringe definition was faulty. (6) When immersion medium index of refraction determinations departed from mean determinations by a significant amount. No photographs were rejected on the basis of computed results. The optical path difference was computed from the evaluated fringe shift (Fig. 1). B = ; A, (A = 0.826 !.L). The thickness (d) in microns, assumed to be equal to the width, was derived by comparison of the measured d with a stage micrometer photograph also taken at 826 nm. Then the index of the outer segment (n,) is given by 6 = [nz - n,]d
8
n, = - _t n,
d
where n, is the index of the immersion medium (Table 1). A computer controlled microdensitometer was used to facilitate the analysis. The instrument consists of an optical image scanner connected to an IBM 360-50 computer. A cathode ray tube serves as a randomprogrammable point source light addressed by the computer on a 8192 x 8192 point grid. The imaged radiant energy passing through each 35 mm data film point is collected at one photomultiplier and the transmitted signal is compared with that of a reference photomultiplier measuring the output of the source alone. The logarithm of the ratio (transmittance) was digitized on a O-63 numerical scale and returned to the computer. The basic point measurement cycle required 20 @sec. A magnified display of a 256 x 256 grid of points above a hxed numerical threshold is shown in Fig. 2. The rod outer segment was seen by the densitometer only as a systematic disturbance in the interference fringe pattern. The fringe separation was measured by scanning across two fringes immediately to the side of the outer segment with a line scan parallel to the cell wall. A linear density array of 200 points was produced and the centers of the two density peaks were computed by forming first moments of the peak shapes. The fringe displacement was determined by scanning across a displaced fringe in the center of the outer segment and scanning in a parallel direction across the undisturbed fringe on each side. The fringe peaks were located by forming hrst moments of the 200 measurements used in each scan. The fringe shift was obtained as the displacement of the center peak in a direction perpendicular to the line joining the two side peaks. The fractional fringe shift of each frame was taken as the mean of the individual quotientsn/b as obtained from ten repeated measurements of each pair taken on 10 separate days. Typical values were about 0.5 fringe and the mean precision of all determinations was 0.33 per cent. Individual precisions are included in Table 2. Measurements of outer segment image width were obtained with an elaborate scheme for producing a maximum-line-of-disturbance signal. The cell edge was detined as that line which had a maximum average transverse density gradient, where positive and negative gradients were separately averaged, then maximized. The length and initial location of a trial edge line were imposed by the operator who controlled the locations of cursor points on a computer-displayed image of the cell fringe pattern (Fig. 2). At each of 50 equally spaced points along this line, a transverse density scan of 11 points was performed, and a least-square fit to the transverse density was used to estimate the density gradient. The 50 transverse density gradient values were combined into separated positive and negative gradient signals. In Fig. 2, one notes that along the lateral border of the outer segment, dispIaced fringes result in alternate positive and negative transverse gradientshence the utility in separating the two signs of gradient signals. As the operator varied the initial lateral displacement and angular orientation of the trial line, the computer repetitively performed the scans, fitted, averaged and displaced the numerical values. The operator oriented the line to approximately maximize the sum of the positive and negative signals. The computer used this line as the starting point in an automated search procedure. The line was stepped in 1” rotations through 5 5” about the initial line. The 11 values of average density form peaks whose centroids were located separately for the positive and negative signals. The line was then reoriented to the optimal angle, and 11 transverse steps of lateral position were made over a range approximately equal to half an outer segment width. The gradient signal values again form peaks whose centroids determine the final ‘best-fit” line location and orientation. This process was repeated for the other lateral border of the outer segment. The final result
FIG. 1. Schematic drawing of a frog rod preparation as viewed through the microscope. Fringe separation, b, and fringe displacement, a, are measured. From these data and the test wavelength (826 run), difference in optical path length, 6, can be computed. Assuming width change is equivalent to diameter change in a cyiindrical rod, path length ditference through the cell and index of refraction of the immersion medium allows a determination of outer segment index of refraction (x2) along the test beam. E-vector was perpendicular to the long axis of the rod in these experiments.
FIG. 2. Polaroid photograph of microdensitometer display during analysis. [facing page 174
B!esching
E?fect on Frog
I75
Otctzr Segment
TABLE 2. DETERVIUATIOM A\D CALCULATIOLS
Specimen
Time
~__ Ffame No. I
Average Average
55B 58D 58E 58H 59B 59A 72E
04018 o-4452 04487 04419 0.4746
_ Prebleach ____0.41 0.23 0.29 0.18 0.25
5’13” 3’43” 2’50” 3’32” 5’31” 5’55” 1’32”
of 5 of 7 5’16” 3’46” 2’54” 3’35” 5’34” 5’58” 4’35”
of 5 of 7
0~4000 04410
0.36 0.15 0.34 0.31 0.18
0.4468
0.4624 0.4830
0.4139 04458 0.4504 04476 0.4728
0.53 0.27 0.19 0.29 0.19
04461
.^ _ Frame No. 4
Average Average
55B 58D 58E 58H 59B 59A 72E of 5 of 7
Frame No. 5
Average Average
5’41” 4’06” 3’17” 3’55” 5’52” 6’20” 4’51”
of 5 of 7 5’44” 4’12” 3’21” 3’58” 5’55” 6’23” 4’54”
1.33061 I.33061 1.33061 1.33000
1.-LO66 1.3934 1.396-! 1.39isl 14052
1.3995
5.21 7.08 6.78 6.70 6.34 5.72 5.10 6.12 6.13
0.06 0.12 0.12 0.27 0.20
5.31 7.05 6.72 6.65 6.28 5.74 5.14 6.38 6.11
0.09 0.13 0.22 0.33 0.19
04053 0.4336 0.4578 04419 O-4782
04038 04469 0.458 1 04440 0.4761
04457
1406’ 1.3928 1.3979 1.3996 1~40610
1.4005
1.32951 1.33061 1.33061 1.33061 1.33000
1.4OS9 1.3938 I .3976 1.3979 14052
1.4006
-Y-77---
Postbleach 04073 04400 0.4535 04424 0.4734
1.32951 1.33061 1.33061 1.33061 1.33000
second bleach 069 0.20 0.22 0.97 0.20
0.60 0.20 0.26 0.37 0.30
04433
55B 58D 58E 58H 59B 59A 72E of 5 of 7
10
04433
55B 58D 58E 58H 59B 59A 72E
Frame No. 6
Average Average
__~____ 5’38” 4’03” 3’14” 3’52” 5’49” 6’17’ 4’48”
0.14 0.15 0.15 0.19
6.13 6.14
0.4486
55B 58D 58E 58H 59B 59A 72E
5.21 7.08 6.81 6.74 6.31 5.12 5.i 1
04424
558 58D 58E 58H 59B 59A 72E
Frame No. 3
Average Average
5’10” 3”O” 7 2’44” 3’29” 5’28” 5’52” 4’29”
of 5 of 7
Frame No. 2
Average Average
-.___
0.35 0.34 0.31 0.35 0.24
5.35 7.16 6.85 6.72 6.37 5.71 5.20 6.49 6.20
0.16 0.11 0.20 0.20 0.18
5.29 7.09 6-83 6.73 6.37 5.71 5.13 646 6.16
0.19 0.14 0.26 0.08 0.12
5.31 7,04 6.85 664 6.37 5.76 5.15 6-44 6-16
0.35 0.21 0.23 0.18 0.08
-______ 1.32951 1.33061 1.33061 I.33061 1.33000
14056 1.3920 1.3968 1.3964 14043
I.3990
1.32951 1.33061 1.33061 1.33061 1.33000
14061 1.3917 1.3976 1.3962 14050
1.3993
1.32951 1.33061 1.33061 1.33061 1.33000
14055 1.3940 1.3974 1.3974 14047
1.3998
176
JAY M. ESOCH, JOHN SCANDRETT AND
5 4
;
FRANC L.
TOBEY, JR.
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FIG. 3. Means of ten microdensitometer readouts of cell width taken on the three photographic frames recorded just before and the three just after a lo-set white light bleach. d is plotted in microns. The arrow gives the time from decapitation to the last photograph before bleach. displayed to the operator was the outer segment taper in degrees and diameter, defined as the spacing between beat-fit centers. For a typical outer segment 10 replications of the width measurement, taken on different days, showed a 0 of O-18 per cent. In order to combine data from several samples, an adjusted time scale had to be established. Each photo-
Time,
set
FIG. 4. Combined means of d for all seven ceils from Fig. 3. It is apparent that exposure to light caused an increase in outer segment width.
177
Weaching Effect on Frog Outer Segment
graph from each preparation was taken at a different time after decapitation, and each bleach was initiated at d&rent moment on each sample. Times for the individual frames were averaged, and these mean times were plotted relative to the average time of onset of the bleacfimg light (e.g. Fig. 4). WhiIe twenty exposures were taken on each sample, only the six taken immediatelybefore and after the bltich were actually anaiyzed instrumentaily. It was found that this procedure reduced judgment errors by the operator who was actively involved in locating reference measurement points on the storage CRT display. Many hours of computer time were required for the analysis of each cell. RESULTS Ofthe many receptors studied, onfy seven met the stringent requirements outtined above for measurement of outer segment width. Two of these had minor imperfections which interfered with determinations of optical path length. Table 2 gives the mean data on each exposure of each sample and the derived quantities. Figure 3 shows the width measurements made on the seven acceptable sets of photographs, and Fig. 4 shows the mean width data. AIthough the observed changes are superimposed upon on going changes due to deterioration of the dying cell fragments (e.g. ENOCH,1967b), it is nevertheless clear that a small increase in width occurred following the bleach. u/dfor most width determinations was about O-2per cent (Table 2). Thus representative vaIues of sigma were about 0.012 p or 120 A, little more than the thickness of a unit membrane. Conside~ng the fact that the measurements were made at 0,826~ and that the numerical aperture of the interference microscope was reduced below optimum, this is a precision well beyond that usually expected in microscopy.
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Difference in optical path length (8) before and after bleach. Mean data from ten independent readouts from each of three frames recorded just before and three just after the IO-see white light bleach. Five of the seven cells from Fig. 3 were used. FIG. 5.
V.I. 1311-M
i78
JAY M.
ENOCH,JOHN
SCAXDRE~
ASD FRANK L. TOBEY, JR.
In those figures referring to individual cells, a common time scale has been plotted, with the actual time of one photograph indicated. The time after decapitation is included in Table 2. Figure 5 shows the optical path length difference of the acceptable specimens. On the basis of these individual records and of the mean data (Fig. 6), it is evident that little or no change occurred in optical path length difference following bleach.
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,, IO Adjusted
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FIG. 6. Combined summary data for five cells. Outer segment diameter and optical path length
axe measured quantities. Outer segment index of refraction (n2) is derived.
Two approaches may be used in evaluating Fig. 6, neither fully satisfactory. The mean of the three values before bleach may be compared with the mean after bleach. This tends to overlook differing secular behaviour in the cell fragments, and treats all variation between individual frames as due to measurement unce~ainty which is not correct. There is remarkably small variance in our analysis techniques. A second alternative is to project a trend from the prebleach to beyond the bleach period. We feel there is too much uncertainty in any apparent trend to warrant this approach. Nevertheless, from simple inspection of Figs. 4 and 6, one can estimate that on an average, bleaching induced an increase in diameter of 2-4 per cent. Sirnilariy, we conclude that there is probably less than a I per cent change in optical path length. Thus the average index of the outer segment must have decreased on the order of 0*1-@2 per cent following a bleach. (2-4 per cent change in index difference.> Taking the measured width to equal the thickness, we obtain a value of about 1400 for she absolute index of refraction of the frog outer segments. This compares reasonably well
Bleaching Effect on Frog Outer Segment
179
with the value of l-411 previously reported by SID.MAN(1957), particularly when one considers the test wavelength difference and the fact that Sidman did not consider postmortem changes or light induced effects. DISCUSSION
Our results indicate that the changes observed here should have very little effect on the waveguide properties of the frog outer segment. For example, the numerical aperture of the fiber, given by ff3 sin a = t/&’ - nlz) is virtually unaltered by bleach. In this equation, n3 is the index of the medium preceeding the fiber optics element, and a is the largest angle of incident light containedby total reflection within the fiber (e.g. ENOCH, 1963). The fact that a limiting numerical aperture exists for fiber optics elements (such as retinal receptors) indicates that a11are directionaiiy sensitive to some degree. An important factor which, in part, describes the transmission capability of a cylindrical waveguide is its cutoff parameter (SNITZEXand OSTERBERG, 1961) p = ;
y’(n*2 - It,*),
where d is the diameter of the waveguide, A is wavelength in vacuum, and nZ and n, are the refractive indexes of the core (outer segment contents) and cladding (interstitial matrix or medium) respectively. The cutoff paramater is simply the product of the waveguide circumference in wavelength units times the numericaf aperture of the guide. Each waveguide modal pattern itself is characterized by a cutoff parameter u,,, which is a particular value of the arg~ent of the Bessel function J_(u) describing the radial dependence of the E(electric) and H(magnetic) vectors of the mode. u,, is that value for which J,(ur/O-56) goes to zero_ Here r is the radial coordinate, and u is a parameter which fixes the scale of the Bessel function relative to the diameter of the core, d. A waveguide may transmit any mode for which
However, this relation does not determine which mode (or modes) is actuafly transmitted in a given case. ENOCH(1963) gives a list of low order modal parameters. Our data indicate that the observed change in d would tend to increasep by 1-3 per cent while the change in n, would tend to decreasep by O-S-2 per cent. Using mean values (5 cells) from Table 2, the resultant value of p increases by O-45%. Changes inp alter the wavelength of cutoff induced transition from one mode to another. A change in modal pattern alters the energy distribution and hence the absorption to some extent. This could lead to local perturbations of the absorption curve which would effect the difference between pre- and post-bleach spectra. Certainly, however, the effects should be minor. We can compute from the above expression for p that changes of the observed magnitude would shift the transition wavelengths on the order of 2-3 nm, which is near the resolution limit of most ~~os~trophotomet~ of visual receptor cells. The difference in optical path length between the frog outer segment and the aqueous surround is primarily due to the high molecular weight protein, phospholipid and lipid constituents present in the highly organized membrane and opsin rich outer segment. On the other hand, the concentration of high molecular weight constituents is low in the aqueous
180
JAY M. ENCH, JOHNSCANDRETT AND FRANKL. TOBEY, JR.
humor, as may be inferred from the index of refraction of the immersion medium which hes close to that of water (Table 1). &RER (1957) derives concentration of solids in individual parts of retinal receptors, using the relationship: C - n2 - no, a
where C is total concentration of solids in mass per unit volume (g/l00 ml), and no is the index of the solvent. The latter may be interchanged with nt, if n, approaches n, (R. BARER, 1957, 1966). a, the specific refraction increment, is treated as a constant for a given ty-pe of biofogical material and has units of volume per unit mass. If n, = n,, 6 = (n2 - n,)d = aCd.
Strictly speaking, this relationship applies to homogeneous cells of uniform thickness (BARER,1966). As indicated below, one must use care in its appii~tion. It is worth consideling briedy the nature of the changes taking place. Let the prebfeach condition be designated by the subscript x and postbleach by y. Our findings can be summarized by 6X= a,, and From above
d y = 1.03d,
aCJX = aGydp,
assuming that width = thickness. It follows that C& = C,( 143dJ
c,
=
l.O3C,
This leads to the conclusion that average concentration of solids, C,, decreased by about 3 per cent. Assuming low index material moves iuto the outer segment, cross sectional area and volume per unit length should increase by 6 per cent, and hence average concentration would decrease by the same amount, neglecting changes in length. Change in length can not be neglected, of course, in estimating total change in volume. The observations could be explained if a 3 per cent increase (say) in diameter were accompanied by a 3 per cent decrease in length. Net volume increase would be 3 per cent resulting in a 3 per cent decrease in refractive index difference or a change just sufEcient to cancel the increased geometrical path. ~~o~~te~y, length was not measured in this study as we were interested primarily in waveguide effects, and a waveguide comparable in length to the outer segment cau be regarded as Snitely long. In any case, the method employed is not suitable for precise length measurements. WEALE(197Ob, 1971~) has suggested that bleaching causes a slight shortening of the outer segment. His estimate ranges between O-02and O-05 pFor a frog outer segment 40 p long, a reduction by 0.05 p would reduce the total volume by only O-1 per cent which is negligible compared to the effects reported here. We have considered another simplified model consistent with the observed data. If bleaching causes water to enter through the ceil membrane, r, but the water remains as a hollow cylinder about the disc core, diameter would increase, but measured difference in
Bleaching Effect on Frog Outer Segment
181
optical path length would not change, since the concentration and thickness of high molecufar weight constituents would not be altered. In this model waveguide properties would not change. We have implicitfy assumed above that the narrow ciiium acts as at least a partial barrier to inflow from the medium via the fractured inner segment. Moreover, in a recent study, CO~IE?J(1971, see his Fig. 2) shows that isolated outer segments from retinas exposed to hypotoic solutions retain their swollen form, suggesting that the cell membrane broken in the process of separation of the fragment acts as if it were healed. ~uwt~nm (I 970) has obtained scanning electron micrographs of rat and monkey retinas follofing intense, long duration exposures to light (indirect ophthalmoscope, for up to 4.5 min). His prints appear to show much greater swelling of outer segments than we have observed, as well as irregularities in the surface and vacuole formation in the Iamellar discs. If these gross dimensional changes are real, one could predict that retinal detachment must accompany such extreme exposures unless there is compensatory shrinkage of the interstitial matrix. The irregularities should result in markedly increased light scatter (MARCUSE, 1969). We have observed nothing resembling these changes in our- experiments. It is clear that more sophisticated models are required to explain the effects of bleaching on receptor outer segments. In particular, the models presented above do not account for all changes in birefringence (WEALE, 1971a,b,c,d) conformational changes (HELLER, 1968) or swefling behavior (COHEN, 1971). Neverthetess, it appears that the effects observed here should not seriously interfere with attempts to characterize the transmission properties of the receptor or the absorption spectra of photolabile pigment in single receptors by microspectrophotometry. .&kno&dgements-Dr. HARRYQUI~LEYand Miss EVA BACHMANN assembled the test data. Mrs. JOAN CRAMER O’HAIRand Mrs. BSWZLYLAWWZNCE performed the final computations on the IBM 360 coupled m~roden~itomcter under Dr. SCAXDRETT’S guidance. Dr. HORN PALERof Zeiss assisted in solving some of the compIex optical problems encountered. The authors wish to thank Dn. R. BARER,B. BECKER, S. E~NTWG,A. COHEN and N. DAW for critically reading this manuscript. REFERENCES ALLEN,R., BRWLT, J. and ZEH, R. (1966).In Advances in Dpticai and Electron Microscopy (&ted by R. BARER and V. Coss~m~), Vol. 1, pp. 81-87.Academic Press. London. BARER,R. (1957). Refractometry and interferometiy of living ce&. J. opt. Sac. Am. 47,545-556, BARER.R. (1966). In Physical Techniques in Biologicd Research (edited by A. POLLI~R), Vol. 3A, pp. 44. Academic Press. New York. COHEN,A. I. (1971). Electron microscopic observations on form changes in photoreceptor outer segments and their saccules in response to osmotic stress. J. ceil &oL 48, 547-565. ENOCH,J. M. (1961).Nature of the transmissionof energy in the retinal receptors. J.opt. sot. A~. 51, 1122-I126.
ENOCH,J. M. 0963). OpticaI properties of retinal receptors. .F. opt. Sot. Am. 53,71-85. ENOCH,3. M. (1%7a). The retina as a fiber optics bundle, Appendix B. In fiber Optics, Pr[nc@/es d Applications (edited by N. S. KAPANY), pp. 372-396. Academic Press, New York. (An updated edition should appear in 1972). ENOCH,J. M. (1967b). Comments on “excitation of waveguide modes in retinal receptors”.J.opt. sot.Am_ 57, 548-549. ENOCH,J. M. and GLSMAW L. (1966). Physical and optical changes in excised retind tissue. Imestne, Oph~~~. 5,208-211.
FELDMAN,E. ad KWUWRA, T. (1968).The retinal pigment epithelium. XV.The damaging effects ofradiant energy. A.M.A. Archs Ophrhal. 80,265-279. mRO% F. (1971). Frog Rhodopsrir in situ: OrientatioMIandSpectraral Changes in the Chromphores oflsofared Rerimf Rod ceik Dissertation. The Johns Hopkins University, Baltimore, Md. HFJLr-WJ. (1968).Structure of visualPigments II. Binding of retinal and conformational changes on light exposurein bovinevisual pigment(Xmax = 500 nm). Biocfremistry 7,2914-292o.
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182
ENOCH,JOHNSCANDREI-~ AND FRAXKL. TOBEY,JR.
J. (1966). Some experiments with a photoelectric ophthalmoscope. In Performance of the Eye or Low Lumenunces (edited by M. BAUMANand J. Vos) pp. 171-181. Exerpta Medica Foundation,
KRMJSKOPF,
Amsterdam. Km, W. (1877). Veranderungen der Stabchen durch Licht. Untersuch, a.d. Physiologischen Inst. d. Univ. Heidelberg, Band 1, Heft 1,409-411 (Cites the work of F. VONHo-). KS, T. (1970). Retinal recovery from exposure to light. Am. J. Ophthaf. 70, 187-198. LATES, A. and L~EBEMAN. P. (1970). Cones of living amphibian eye: selective staining. Science, N. Y. 168, 1475-1476. MARCUS~, D. (1969). Mode conversion caused by surface imperfections of a dielectric slab waveguide. Bell
System Tech. J. 48,3187-321X OHZU, H. and ENOCH,J. M. (1972). Optical modulation by the isolated human fovea. Vision Res. 12,
245-25 1. OIUU, H.. ENOCH,J. and Q’HAIR,J. (1972). Optical modulation by the isolated retina and retinal receptors. vision Res. 12,231-244. POLE,R. et al. (1972). Topical Meeting on Integrated Optics-Guided Waves, Materials and Devices. Las Vegas, Nev. Feb. 7-10, 1972. Optical Society of America, Washington, D.C. &MAN, R. (1957). The structure and concentration of solids in photoreceptor ceils studied by refractometry and interference microscopy. J. biophys. biochem. Cytol. 3, 15-20. Sm, E. and OSrEmERG, H. (1961). Optical dielectric waveguide modes in the visible spectrum. J. opt. Sot. Am. 51,499-505. SNYDER,A. and RICHMO~-D, P. (1972). Light absorption in visual photoreceptors. Science, N. Y. (submitted for publication). WASHBURN, E. (editor) 1930. ZntemationaICritical Tables of Numerical Data Physics, Chemistry and Technology. (1st edition), Vol. 7, p. 13. McGraw-Hill, New York. Wute, R. (1964). Comparisons of reactions of human and rabbit fundi to photopic exposures. J. opt. Sot. Am. 54,120-126.
WEALE,R. A. (1965). High light intensities and photo-chemical reactions of human visual pigments in situ. In Symposiu on Quunt. Biol. Vol. 30, pp. 335-343. Cold Spring Harbor Symp. Quant. Biol., New York. WHALE,R. A. (197Oa).Optical properties of photoreceptors. Br. med. Bull. 26,134-136, plate 9. WEALE,R. A. (197Ob).How the birefringence of vertebrate rods is affected by light. J. Physiol., Lond. 210, 28-29P. W~ALJ$R. A. (1971a). On the linear dichroism of frog rods. Vision Res. 11,1373-1385 (Appendix by R. W* SMlTH). WEALE,R. A. (1971b). Rod birefringence and light. VisionRes. 11, 1387-1393. WEALE,R. A. (1971~). On the birefringence of rods and cones. Pmers Arch. ges. Physiol. 329,244-257. Wg,ua, R. A. (1971d). Photolysis and birefringence of frog rods. Experientia 27,403. WOLKEN, J. (1963). Structure and molecular organization of retinal photoreceptors. J. opt. Sot. Am. 53, I-19.
Ah&act-An i&a-red interference microscope has been developed to study the effects of a white light bleach on physical properties of single receptors. The width and optical path length was measured for tie& ftog rod outer segments lying on their sides before and just after a major bleach. The data presented were recorded some seconds after the bleach. Use of a computer controlled microdensitometer allowed determination of these parameters with precision well beyond normaI resolution limits. Following light exposure, receptor width increased between 2-4 per cent and optical path length difference remained virtually unchanged. This suggests that a small decrease in index of refraction occmred in the receptor outer segmentat the wavelength used (826 nm).
R&umGQn a construit up microscope interferentiel infrarouge pour I’etude des effets de la lumi&e blanche sur la prop&& physiques de.s r&epteurs isoiis. On mesure la largeur et le trajet optique de segments extemesfraisdeb&onnetsde grenouilles,couch&ur leursc&&,avant et juste apr&s une d&oloration intense. Lea don&es etaient enregistrees quelques secondes aprts Ia d6coIoration. Un microdensidom&re contr~le par une cabdatrice permetde d&miner ces parametres avec une pr&ision largement sup&ieure aux limitee normales de r&solution. Apt& exposition a la lumiere, la largeur du recepteur augmente de 2 a 4 pour cent et le trajet optique reste pratiquement constant. Cela suggere une petite diminution de l’indice da refraction dans le segment exteme du r&epteur pour la longueur d’onde employ&z (826 nm).
Bleaching Effect on Frog Outer Segment
ZusaMnentaswng-Urn
! 83
die Effekte zu untenuchen, wenn man mit weissem Licht die natiir-
lichen Bestandteile von einzelnen Rezeptoren bleicht, wurde ein Infrarot-Interferenzmikroskop entwickelt. Gemessen wurden die Breite und die opt&he Wegllnge von aug der Seite liegenden Susseren Froschstibchensegmenten vor und nach einer grijsseren Bleichung. Die sich dabei ergebenden Messwerte wurden einige Sekunden nach der Bleichung aufgezeichnet. Durch
Verwendung eines rechnergesteuerten Mikrodensitometers konnten diese Parameter mit einer Genauigkeit weit unter der Gblichen AtiBsung bestimmt werden. Nach der Lichteinstrahlung verbreiterte sich der Rezeptor um Z-4 Prozent, wshrend die opt&he WeglrIngendifferenz unvedndert blieb. Das Itit vennuten, dass die Brechzahl im Busseren Rezeptorsequent bei der verwendeten Wellenllnge von 826 nm etwas kleiner wird.
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