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Retinal densitometer with the size of a fundus camera
0042#89/89
Vision Res. Vol. 29, No. 3, pp. 369-374, 1989 printed in Great Britain.All rightsreserved
S3.00 + 0.00
copyright Q 1989 FcrSamon Ptw p
TECHNICAL NOTE RETINAL
DENSITOMETER WITH THE SIZE OF A FUNDUS CAMERA DIRK VAN NORREN'** and JAN VAN DE KBM~’
‘Royal Ne~erl~ds
Eye Hospital, F.C. Dondersstraat 65,3572 JE Utrecht and 21nstitutefor perception TNO, Kampweg 5, Soesterberg, The Ne~~l~~ (Received 11 My 1988; in reuisedform I1 Augurt 1988)
Ahatraet-This paper describes a small, user-friendly fundus reflection densitometer. All optics and part of the electronics are contained in a box with the sixe of a fundus camera. A personal computer is used for control and on-line display of output. A sin#e 30 W halogen tamp provides bieaching and measuring light. A chopper wheel generates 24 light pulses in 100 msec time frames: 16 pulses of measuring light at different wavelengths covering the spectrum, four pulses of bleaching light (optionally), and four dark pulses for assessing the dark current of the photomuhiplier. The fundus can be viewed when the bleaching light is on. The measuring field has four widths ranging from 1.6 to 5.4 deg; the bleaching light is Bxed at 25 deg. A fixation aid may be positioned anywhere in the bleaching field. A microprocessor sorts the quanta, detected by the photom~tiplier after reflection from the fundw, in 16 channels labeled with wavelength notation. Real-time changes in spectral reflection can be viewed on a monitor. Due to optimal design of entrance and exit pupils fovea1 density dil%erences of up to 0.5 were recorded in human subjects. This is higher than ever reported before with retinal densitometry. Visual pigments Densitometry Difference spectrum
Cones
Rods
INTRODUCMON
About 30 years ago Rushton (1956) and Weale ( 1957)developed techniques to measure changes in the density of visual pigments in the living eye. In a limited number of laboratories this technique has been used to explore the kinetics and spectral characteristics of visual pigments in oh. A substantial barrier for more widespread use, in particufar in a clinical setting, is the problem of developing the complex and costly apparatus (Hood and Rushton, 1971;Ripps and Snapper, 1974). We experienced that, once such a barrier is surmounted (van Norren and van de Kraats, 1981), profitable use of densitometry could be made in studying pigment kinetics in healthy and diseased retinas (e.g. van Meel and van Norren, 1983, 1986; Keunen et al., 1987, 1988; Coolen and van Norren, 1988). In this paper we describe the results of our efforts to develop a new densitometer. We set the following aims.
Pigment regeneration
Time constant
-The apparatus should yield spectral information to evaluate possible changes in relative contribution of different visual pigments, and to assess the distorting influence of bleach products (Ripps et al., 1981). -The performance of the apparatus should be such that the mean fovea1 density in healthy subjects is at least 0.3. Since densitometers are not likely to become co~e~ially available in the near future, the design principles outlined in this paper might be of help to those wishing to construct one.
An optical schematic is given in Fig. 1A. The optical part of the densitometer is confined to a box of approx. 50 x 40 x 10cm3. Light source for both bleaching and measuring beam is a 30 W halogen lamp. The rne~~ng beam is relayed by a set of two lenses, -The apparatus should be user-friendly: small, focussed, again relayed and, at the next focus, easy to operate, with output available on-line reflected towards the eye by a small mirror (M). and digital storage of data. The bleaching beam follows a similar path, and 369
370
Technical Note
observer
bleach
10ms
ID
Fig. 1. (A) Scheme of the optics, approximately to scale. L = lens, R = planes conjugate with retina, M = mirror, PM = photomultiplier. For additional details, see text. (B) Sequence of pulses in measuring and bleaching light paths after the chopper wheel. Dashed pulse in the “measure” beam indicates that at least one hole in the chopper wheel should be blocked for assessing the dark current of the photomultiplier. In the present apparatus four holes are blocked. (C) Location of entrance and exit pupil of the optical system in the plane of the subject’s pupil.
is, between the second set of relay lenses, combined with the measuring beam through a beam splitter. A chopper wheel in the first foci allows the measuring beam to pass at moments that the bleaching beam is blocked (note that these beams pass at different radii), and vice versa. At the radius of the measuring beam the wheel has 20 holes, 16 are fit with an interference filter (effective bandwith 10-14 nm), and 4 are blocked to assess the output of the photomultiplier (PM, see later) in the dark. The sequence of light pulses after the chopper is schematically depicted in Fig. lb. A circular diaphragm (Rl) in the measuring beam between the second set of relay lenses is
focussed at the retina. Four field widths are available (2.3, 3.0,4.5 and 6.9 deg). The bleaching beam has a fixed 25 deg diaphragm with cross hairs for fixation purposes (R2). To reduce the possibility of photochemical damage to the retina by blue light (Ham et al., 1976), this diaphragm has a yellow filter with a sharp cut-off at 5OOnm (Kodak Wratten 8). The bleaching beam is switched off by quickly replacing the circular diaphragm with a pinhole with a blue filter (blue, because the short wavelength sensitive cone system is not much affected by the bleaching light). The blue spot of light becomes visible within a few seconds after switching off the yellow bleach and enables
fixation dting dark adaptation. The whole contraption can be moved laterally t0 allow for eccentric presentation of the measuring light. The exit lens of the densitometer, an aspherical ophthalmoscopic lens with antireflection Coating (Nikon, 23 D), images the lamp filament in the plane of the subject’s pupil (Fig. lC), The reflected fundus light emerging from the whole pupil is refocussed, by the same lens, at the plane of mirror M. A diaphragm (not shown) selects from that image a semiCircular part for further processing (Fig. 1C). Conjugate with the 20 holes at a large radius, the chopper wheel has 20 holes at an intermediate radius through which the measuring light reflected by the fundus passes. This light then passes through a lens and an adjustable diaphragm (R3), conjugate with the retina. The latter defines the diameter of the retinal area from which light is collected by the PM. TO allow for imperfect imaging, this diameter is adjusted to be about 25% smaller than that of the ill~in~ted field (available are fields of 1,6, 2.4, 3.5 or 5.4deg). Unnecessary large illuminated fields increase unwanted stray-light. At the intermediate radius the chopper has four mirrors to deflect the fundus reflected bleach light to an observer’s eye, The PM is thus protected against light of relatively high intensity, and the operator may view the fundus through an eye piece, aided by cross hairs indicating the center of the measuring field (R4). The bleaching light flickers at a frequency of 40 I-Iz, but neither subjects, nor observers reported this as uncomfortable. The maximum retinal illumination by the bleaching light is 5.6 log phot. td, which happened to be also 5.6 log Scot. td. To limit bleaching by the measuring light, neutral density filters were placed on the interference filters such that the time integrated output of all filters together was 880 phot. td (2350 Scot. td). The latter level was higher than we wished, but unavoidable to obtain a reasonable signal to noise ratio in the short wavelength part of the spectrum, We opted for an unequal division of light over the spectrum, in the sense that at the two wavelengths that are displayed as density traces in the standard cone and rod programs (554 and 515 nm, see later) a slightly better signal to noise ratio was obtained than at other wavelengths. This was to aid judgement of the length of dark adaptation period during measurements (see Results and Discussion). The densitometer has a fixed focus. Only for
an emmetropic eye optimum focus and tent&on of stimulus field and the field seen by the photomultiplier is achieved. Yet, due to tie large depth of focus and the overlap of s&ulus and PM field, reliable measurements
are possible up to eight diopters of ametropia.
A Motorola 6809 microprocessor on a board in the optics box controls chopper speed and shutter position, and Teeeives input from the photon counter. Once a second, the microprocessor sends the number of counts collected into 20 channels corresponding to 20 positions in the filter wheel, to a host computer, through a standard RS 232 Connector. In the host camputer, in our case an Atari 1040 ST, data of 16 channels are labeled with wavelength and time information, and Corrected for the dark output of the PM. The latter consists of counts generated by stray-light in the apparatus, room light and by other causes. All data are stored on disk.
Half an hour before a measurement the subject’s eye is instilled with one or two drops of a mild mydriatic to widen the pupil. A minimum width of 2-5 mm is required to harbour entranCe and exit pupils, but a generous margin prevents blockage ‘of light through instabilities in eye position, Immobilisation of the subject’s head is achieved by a bite board (impression compound on a sterilized holder) and temple pads. The operator can adjust the densitometer, just like a fundus Camera, in three dimensions. The operator may choose from a menu “standard cone”, “standard rod” or “experimental program”. In the standard programs timing and wavelengths displayed on the Screen are, to a large extent, pre-programmed. RESULTS
AND DISCUSSION
Samples of the information displayed on screen after completion of standard cone and rod measurements are given in Fig, 2A and B. One trace (plusses) represents the number of counts (output of the PM Corrected for dark Counts) as a function of time for 730 nm. Each data point is the mean of a lo-set interval. The wavelength of 73Onm is not appreciably absorbed by the visual pigments so that the stability of the trace serves as a Check on the reliability of a measurement. In a perfect measurement the trace is a straight line showing
372
Technical Note
time Imin)
0.L
I
1
I
8
12
16
t 20
time
IminI
t
i
0.1 --f
0
’ I
2L
26
32
36
40
Fig. 2. (A) Example of output available on screen after completion of a standard cone program. only the frame and its lettering were redrawn for clarity purposes. Subject was a 38.yr old male, O.D. with 1.5 D of myopia. Measuring field (seen by PM) was 2.4 deg, directed at the fovea. Plusses represent reflected light at 730 nm, crosses at 419 nm. Density, the logarithm of the ratio of the PM output at 730 and 551 nm, corrected for dark output, is represented by open squares. Bleaching light was on from 0 to 2 and from IO to 12 min. Light off is indicated by vertical dashed line, light on by dotted line. The density trace shows regeneration of visual pigment after light off, and rapid phototysis after light on. A horizontal tine, fit through the data points between 0 and 2 min serves as a check for return of the trace in the second bleach period. The two lines of 1min each, drawn above the abscissa in the bleach periods, encompass the data points used for calculation of the mean density in fully bleached condition; the 2 min fine at the end of the dark period defines the data points for the dark adaptedcondition.The latter data are used for off-line calculation of density differences and time constants of regeneration at each wavelength. The trace may be compared to the one in Fig. 6 of van Norren and van de Kraats (1981) with a density difference of 0.33, which was from the same eye (author JK). (B) Same as (A) for standard rod program. Subject was a 45yr old male, O.D. with 6.5 D of myopia. Density trace refers to 515 nm. Measuring field was 5.4 deg, at a locus in the temporal retina at 16 deg eccentricity on the horizontal meridian. The bleaching sequence was 3 min on, 32 min off, 5 on. The subject was allowed to lean back after 13 min in the dark and resumed position 5 min before light on, indicated by lapses in the traces and line below abscissa.
no influence of the on- or offset of the bleaching light and no drifts due to improper fixation. The second trace (open squares), which we termed “density”, is the logarithm of the ratio of the number of counts at the deep red wavelength and the counts at 551 nm (515 nm in the rod program). The third trace (crosses) represents
the number of counts at 419 nm. We chose to display this wavelength because at short wavelengths the reflection factor of the eye is, in principle, very small (van Norren and Tiemeyer, 1986), hence the count number is a sensitive indicator for specular reflections at layers anterior to the receptors, like inner limiting
313
Technical Note 0
I
350
._ 1
LOO
1
LSO
1
500
’
550
*
600
’
650
0
700
’
750
wavelength Inm!
350
LOO
I.50
500
550
600
650
700
750
wovelengih~nml
Fig. 3. (A) Difference spectrum obtained in the fovea1 measurement of Fig. 2A. For comparison absorption spectra of middle (peak at 531 nm) and long wavelength sensitive cones (561 nm) with peak densities of 0.4 (Baylor et al., 1987) are drawn. (B) Time constants of dark adaptation for the fovea1 measurement. Because of low density differences, noise increases at the ends of the spectrum. Below density 0.04 no time constants is calculated. (C) Difference spectrum obtained in the peripheral measurement of Fig. 2B. For comparison a Dartnall nomogram with peak at 492 nm is drawn. (D) Time constant obtained in the peripheral measurement.
lens and cornea. After the start of the standard cone program, the bleaching light is on for 2 min, then off for maximally 15 min, and again on for 2 or 3 min (Fig. 2A). The second bleach allows an additional check for the stability of the measurement, because the trace should return to its previous position during bleach on. In normal subjects the operator generally terminates the dark period after 8 min. In the example of Fig. 2A (right eye of author JK) the density difference between the bleached and dark adapted condition is 0.41. The fovea1 density difference for 17 subjects aged between 20 and 45 yr ranged between 0.35 and 0.49, with a mean of 0.42 and standard deviation of 0.04. Most likely, the increase of the mean with respect to earlier mean data recorded with our previous densitometer (around 0.30) is due to the limited size (now within 2.0 mm, previously 3.2 mm) and the proximity of entrance and exit pupil, which allows positioning close to the maximum of the StilesXrawford effect. Thus, longer pathlengths of the measuring light are obtained in cone outer segments (van Blokland and van Norren, 1986). A disadvantage of the small entrance and exit pupils is that positioning becomes very critical. Highest density differences were obtained with the following criteria. First, symmetric superficial retinal reflexes around the fovea; second, focussing at membrane,
anterior surface of cristalline lens, and third, low counts at 419 nm and high counts at middle wavelengths. Results of a standard rod measurement program (right eye of author DN) are presented in Fig. 2B. The bleaching sequence is 3 min on, maximally 32 min off, and 5 min on. Since a measurement of such length is difficult to endure, the subject is generally allowed to lean back after 10-15 min in the dark, and to resume position about 5 minutes before light on (visible as lapses in the traces of Fig. 2B). Data were obtained at 16 deg eccentricity on the horizontal meridian, juxtapose the blind spot. The measuring field was 5.4deg, Resulting in much higher count numbers and hence, better signal to noise ratio than in the fovea1 measurements where field size was 2.4deg (note the differences in density scale between Figs 2A and B). The maximum density difference was 0.16. After completion of a measurement, a difference spectrum is available with baseline (fully bleached) defined as a linear interpolation between the mean level 60 set before light off and the last 60 set of the measurement. The dark adapted level is the mean of the last 60 set before light on. The spectra obtained in the sessions of Fig. 2 are displayed in Fig. 3. With fovea1 measuring field the spectrum peaks near 550 nm, indicative for cone pigments. For
374
Technical Note
comparison middle (peak at 531 nm) and long (561 nm) wavelength sensitive cone absorption spectra (Baylor et al., 1987) are drawn through the data obtained (Fig. 3A). A proper comparison, which falls outside the scope of the present paper, would require an analysis that takes stray light, ratio of middle to long wavelength sensitive cones, and bleach products into account. Through the spectrum obtained at 16deg eccentricity of Dartnall nomogram with peak at 492nm is drawn (Fig. 3C). As was already noted by Rushton in 1956 the rod difference spectrum shows a substantial red shift. Also available is a curve fitting program which yields the time constants (l/e value) of regeneration for all wavelengths under the assumption that regeneration follows an exponential decay (which holds only to first approximation, cf. Smith et al., 1983; Coolen and van Norren, 1988). Data obtained with this program are plotted in Fig. 3B and D. Time constants of recovery are about 100 set with fovea1 fixation, and about 300 set at 16 deg eccentricity, corroborating the shift from cone to rod pigment. At the extremes of the spectrum low density differences prevent calculation of time constants. In conclusion, we built a retinal densitometer which is easy to use and suited for the study of kinetics and spectral characteristics of visual pigments, Fovea1 density differences were higher than ever recorded. Rcknawledgemnts-We thank C. D. van der Linden for his ~ont~bution in developing mechanical components, and G. 3. van Meel MD, J. E. E. Keunen MD and E. Bertelh MD for helpful discussions.
REFERENCES
BaylorD. A., Nunn B. J. and SchnapfJ. L. (1987) Spectral sensitivity of cones of the monkey Macnca .~seic~arjs. J. Fhysiol. Land. 390, 145-160.
van Blokland and van Norren D. (1986) Intensity and polarisation of light scattered at small angles from the human fovea. Vision Res. 26, 485-494. Coolen A. C. C. and van Norren D. (1988) kinetics of human cone pigments explained with a Rushton-Henry model. Biol. Cyberne?. 58, 123-128. Ham W. T., Mueller H. A. and Shney D. H. (1976) Retinal sensitivity to damage from short wavelength light. Nature Land. 260, 153-155.
Hood C. and Rushton W. A. H. (1971) The Florida retinal densitometer. J. Physiot. Lond. 217, 501-511. Keunen J. E. E., van Norren D. and van MeeI G. .I. (1987) Density of cone pigments at aider age. Invest. Qpht~~. visual Sci. 28, 985-99 I. Keunen J. E. E., van Meel G. J. and van Norren D. (1988) Rod densitometry in congenital stationary night blindness. Appl. Upr. 27, 1050-1056. van Meel G. J. and van Norren D. (1983) Fovea1 densitometry in Retinitis Pigmentosa. Invesr. Ophthul. u&al Sci. 24, 1123-l 130.
van Meei G. J. and van Norren D. (1986) Fovea1 densitometry as a diagnostic technique in Stargardt’s disease. Am. J. Ophthal.
102, 353-362.
van Norren D. and van de Kraats J. (1981) A continuously recording retinal densitometer. Vision Res. 21, 897-905. van Norren D. and Tiemeyer L. F. (1986) Spectral reflectance of the human eye. V&an Res. 26, 313-320. Ripps H. and Snapper A. G. (1974) Computer analysis of photochemical changes in the retina. Comput. Biol. Med. 4, 107-122.
Ripps H., Mehaffy III L. and Siegel I. M. (1981) Rh~opsin kinetics in the cat retina. J. gen. Physiof. 77, 31’7-334. Rushton W. A. H. (1956) The difference spectrum and photo~nsit~vity of rhodopsin in the living human eye. J. Physiol. Lond. 134, 1 b-29.
Smith V. C., Pokomy J. and van Norren D. (1983) Densitometric measurement of human cone photopigment kinetics. Vision Res. 23 517.--524. Weale R. A. (1957) Observations on photochemical reactions in living eyes. Br. J. Up~thaI. 41, 461-474.
Vision Res. Vol. 29, No. 3, pp. 369-374, 1989 printed in Great Britain.All rightsreserved
S3.00 + 0.00
copyright Q 1989 FcrSamon Ptw p
TECHNICAL NOTE RETINAL
DENSITOMETER WITH THE SIZE OF A FUNDUS CAMERA DIRK VAN NORREN'** and JAN VAN DE KBM~’
‘Royal Ne~erl~ds
Eye Hospital, F.C. Dondersstraat 65,3572 JE Utrecht and 21nstitutefor perception TNO, Kampweg 5, Soesterberg, The Ne~~l~~ (Received 11 My 1988; in reuisedform I1 Augurt 1988)
Ahatraet-This paper describes a small, user-friendly fundus reflection densitometer. All optics and part of the electronics are contained in a box with the sixe of a fundus camera. A personal computer is used for control and on-line display of output. A sin#e 30 W halogen tamp provides bieaching and measuring light. A chopper wheel generates 24 light pulses in 100 msec time frames: 16 pulses of measuring light at different wavelengths covering the spectrum, four pulses of bleaching light (optionally), and four dark pulses for assessing the dark current of the photomuhiplier. The fundus can be viewed when the bleaching light is on. The measuring field has four widths ranging from 1.6 to 5.4 deg; the bleaching light is Bxed at 25 deg. A fixation aid may be positioned anywhere in the bleaching field. A microprocessor sorts the quanta, detected by the photom~tiplier after reflection from the fundw, in 16 channels labeled with wavelength notation. Real-time changes in spectral reflection can be viewed on a monitor. Due to optimal design of entrance and exit pupils fovea1 density dil%erences of up to 0.5 were recorded in human subjects. This is higher than ever reported before with retinal densitometry. Visual pigments Densitometry Difference spectrum
Cones
Rods
INTRODUCMON
About 30 years ago Rushton (1956) and Weale ( 1957)developed techniques to measure changes in the density of visual pigments in the living eye. In a limited number of laboratories this technique has been used to explore the kinetics and spectral characteristics of visual pigments in oh. A substantial barrier for more widespread use, in particufar in a clinical setting, is the problem of developing the complex and costly apparatus (Hood and Rushton, 1971;Ripps and Snapper, 1974). We experienced that, once such a barrier is surmounted (van Norren and van de Kraats, 1981), profitable use of densitometry could be made in studying pigment kinetics in healthy and diseased retinas (e.g. van Meel and van Norren, 1983, 1986; Keunen et al., 1987, 1988; Coolen and van Norren, 1988). In this paper we describe the results of our efforts to develop a new densitometer. We set the following aims.
Pigment regeneration
Time constant
-The apparatus should yield spectral information to evaluate possible changes in relative contribution of different visual pigments, and to assess the distorting influence of bleach products (Ripps et al., 1981). -The performance of the apparatus should be such that the mean fovea1 density in healthy subjects is at least 0.3. Since densitometers are not likely to become co~e~ially available in the near future, the design principles outlined in this paper might be of help to those wishing to construct one.
An optical schematic is given in Fig. 1A. The optical part of the densitometer is confined to a box of approx. 50 x 40 x 10cm3. Light source for both bleaching and measuring beam is a 30 W halogen lamp. The rne~~ng beam is relayed by a set of two lenses, -The apparatus should be user-friendly: small, focussed, again relayed and, at the next focus, easy to operate, with output available on-line reflected towards the eye by a small mirror (M). and digital storage of data. The bleaching beam follows a similar path, and 369
370
Technical Note
observer
bleach
10ms
ID
Fig. 1. (A) Scheme of the optics, approximately to scale. L = lens, R = planes conjugate with retina, M = mirror, PM = photomultiplier. For additional details, see text. (B) Sequence of pulses in measuring and bleaching light paths after the chopper wheel. Dashed pulse in the “measure” beam indicates that at least one hole in the chopper wheel should be blocked for assessing the dark current of the photomultiplier. In the present apparatus four holes are blocked. (C) Location of entrance and exit pupil of the optical system in the plane of the subject’s pupil.
is, between the second set of relay lenses, combined with the measuring beam through a beam splitter. A chopper wheel in the first foci allows the measuring beam to pass at moments that the bleaching beam is blocked (note that these beams pass at different radii), and vice versa. At the radius of the measuring beam the wheel has 20 holes, 16 are fit with an interference filter (effective bandwith 10-14 nm), and 4 are blocked to assess the output of the photomultiplier (PM, see later) in the dark. The sequence of light pulses after the chopper is schematically depicted in Fig. lb. A circular diaphragm (Rl) in the measuring beam between the second set of relay lenses is
focussed at the retina. Four field widths are available (2.3, 3.0,4.5 and 6.9 deg). The bleaching beam has a fixed 25 deg diaphragm with cross hairs for fixation purposes (R2). To reduce the possibility of photochemical damage to the retina by blue light (Ham et al., 1976), this diaphragm has a yellow filter with a sharp cut-off at 5OOnm (Kodak Wratten 8). The bleaching beam is switched off by quickly replacing the circular diaphragm with a pinhole with a blue filter (blue, because the short wavelength sensitive cone system is not much affected by the bleaching light). The blue spot of light becomes visible within a few seconds after switching off the yellow bleach and enables
fixation dting dark adaptation. The whole contraption can be moved laterally t0 allow for eccentric presentation of the measuring light. The exit lens of the densitometer, an aspherical ophthalmoscopic lens with antireflection Coating (Nikon, 23 D), images the lamp filament in the plane of the subject’s pupil (Fig. lC), The reflected fundus light emerging from the whole pupil is refocussed, by the same lens, at the plane of mirror M. A diaphragm (not shown) selects from that image a semiCircular part for further processing (Fig. 1C). Conjugate with the 20 holes at a large radius, the chopper wheel has 20 holes at an intermediate radius through which the measuring light reflected by the fundus passes. This light then passes through a lens and an adjustable diaphragm (R3), conjugate with the retina. The latter defines the diameter of the retinal area from which light is collected by the PM. TO allow for imperfect imaging, this diameter is adjusted to be about 25% smaller than that of the ill~in~ted field (available are fields of 1,6, 2.4, 3.5 or 5.4deg). Unnecessary large illuminated fields increase unwanted stray-light. At the intermediate radius the chopper has four mirrors to deflect the fundus reflected bleach light to an observer’s eye, The PM is thus protected against light of relatively high intensity, and the operator may view the fundus through an eye piece, aided by cross hairs indicating the center of the measuring field (R4). The bleaching light flickers at a frequency of 40 I-Iz, but neither subjects, nor observers reported this as uncomfortable. The maximum retinal illumination by the bleaching light is 5.6 log phot. td, which happened to be also 5.6 log Scot. td. To limit bleaching by the measuring light, neutral density filters were placed on the interference filters such that the time integrated output of all filters together was 880 phot. td (2350 Scot. td). The latter level was higher than we wished, but unavoidable to obtain a reasonable signal to noise ratio in the short wavelength part of the spectrum, We opted for an unequal division of light over the spectrum, in the sense that at the two wavelengths that are displayed as density traces in the standard cone and rod programs (554 and 515 nm, see later) a slightly better signal to noise ratio was obtained than at other wavelengths. This was to aid judgement of the length of dark adaptation period during measurements (see Results and Discussion). The densitometer has a fixed focus. Only for
an emmetropic eye optimum focus and tent&on of stimulus field and the field seen by the photomultiplier is achieved. Yet, due to tie large depth of focus and the overlap of s&ulus and PM field, reliable measurements
are possible up to eight diopters of ametropia.
A Motorola 6809 microprocessor on a board in the optics box controls chopper speed and shutter position, and Teeeives input from the photon counter. Once a second, the microprocessor sends the number of counts collected into 20 channels corresponding to 20 positions in the filter wheel, to a host computer, through a standard RS 232 Connector. In the host camputer, in our case an Atari 1040 ST, data of 16 channels are labeled with wavelength and time information, and Corrected for the dark output of the PM. The latter consists of counts generated by stray-light in the apparatus, room light and by other causes. All data are stored on disk.
Half an hour before a measurement the subject’s eye is instilled with one or two drops of a mild mydriatic to widen the pupil. A minimum width of 2-5 mm is required to harbour entranCe and exit pupils, but a generous margin prevents blockage ‘of light through instabilities in eye position, Immobilisation of the subject’s head is achieved by a bite board (impression compound on a sterilized holder) and temple pads. The operator can adjust the densitometer, just like a fundus Camera, in three dimensions. The operator may choose from a menu “standard cone”, “standard rod” or “experimental program”. In the standard programs timing and wavelengths displayed on the Screen are, to a large extent, pre-programmed. RESULTS
AND DISCUSSION
Samples of the information displayed on screen after completion of standard cone and rod measurements are given in Fig, 2A and B. One trace (plusses) represents the number of counts (output of the PM Corrected for dark Counts) as a function of time for 730 nm. Each data point is the mean of a lo-set interval. The wavelength of 73Onm is not appreciably absorbed by the visual pigments so that the stability of the trace serves as a Check on the reliability of a measurement. In a perfect measurement the trace is a straight line showing
372
Technical Note
time Imin)
0.L
I
1
I
8
12
16
t 20
time
IminI
t
i
0.1 --f
0
’ I
2L
26
32
36
40
Fig. 2. (A) Example of output available on screen after completion of a standard cone program. only the frame and its lettering were redrawn for clarity purposes. Subject was a 38.yr old male, O.D. with 1.5 D of myopia. Measuring field (seen by PM) was 2.4 deg, directed at the fovea. Plusses represent reflected light at 730 nm, crosses at 419 nm. Density, the logarithm of the ratio of the PM output at 730 and 551 nm, corrected for dark output, is represented by open squares. Bleaching light was on from 0 to 2 and from IO to 12 min. Light off is indicated by vertical dashed line, light on by dotted line. The density trace shows regeneration of visual pigment after light off, and rapid phototysis after light on. A horizontal tine, fit through the data points between 0 and 2 min serves as a check for return of the trace in the second bleach period. The two lines of 1min each, drawn above the abscissa in the bleach periods, encompass the data points used for calculation of the mean density in fully bleached condition; the 2 min fine at the end of the dark period defines the data points for the dark adaptedcondition.The latter data are used for off-line calculation of density differences and time constants of regeneration at each wavelength. The trace may be compared to the one in Fig. 6 of van Norren and van de Kraats (1981) with a density difference of 0.33, which was from the same eye (author JK). (B) Same as (A) for standard rod program. Subject was a 45yr old male, O.D. with 6.5 D of myopia. Density trace refers to 515 nm. Measuring field was 5.4 deg, at a locus in the temporal retina at 16 deg eccentricity on the horizontal meridian. The bleaching sequence was 3 min on, 32 min off, 5 on. The subject was allowed to lean back after 13 min in the dark and resumed position 5 min before light on, indicated by lapses in the traces and line below abscissa.
no influence of the on- or offset of the bleaching light and no drifts due to improper fixation. The second trace (open squares), which we termed “density”, is the logarithm of the ratio of the number of counts at the deep red wavelength and the counts at 551 nm (515 nm in the rod program). The third trace (crosses) represents
the number of counts at 419 nm. We chose to display this wavelength because at short wavelengths the reflection factor of the eye is, in principle, very small (van Norren and Tiemeyer, 1986), hence the count number is a sensitive indicator for specular reflections at layers anterior to the receptors, like inner limiting
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Fig. 3. (A) Difference spectrum obtained in the fovea1 measurement of Fig. 2A. For comparison absorption spectra of middle (peak at 531 nm) and long wavelength sensitive cones (561 nm) with peak densities of 0.4 (Baylor et al., 1987) are drawn. (B) Time constants of dark adaptation for the fovea1 measurement. Because of low density differences, noise increases at the ends of the spectrum. Below density 0.04 no time constants is calculated. (C) Difference spectrum obtained in the peripheral measurement of Fig. 2B. For comparison a Dartnall nomogram with peak at 492 nm is drawn. (D) Time constant obtained in the peripheral measurement.
lens and cornea. After the start of the standard cone program, the bleaching light is on for 2 min, then off for maximally 15 min, and again on for 2 or 3 min (Fig. 2A). The second bleach allows an additional check for the stability of the measurement, because the trace should return to its previous position during bleach on. In normal subjects the operator generally terminates the dark period after 8 min. In the example of Fig. 2A (right eye of author JK) the density difference between the bleached and dark adapted condition is 0.41. The fovea1 density difference for 17 subjects aged between 20 and 45 yr ranged between 0.35 and 0.49, with a mean of 0.42 and standard deviation of 0.04. Most likely, the increase of the mean with respect to earlier mean data recorded with our previous densitometer (around 0.30) is due to the limited size (now within 2.0 mm, previously 3.2 mm) and the proximity of entrance and exit pupil, which allows positioning close to the maximum of the StilesXrawford effect. Thus, longer pathlengths of the measuring light are obtained in cone outer segments (van Blokland and van Norren, 1986). A disadvantage of the small entrance and exit pupils is that positioning becomes very critical. Highest density differences were obtained with the following criteria. First, symmetric superficial retinal reflexes around the fovea; second, focussing at membrane,
anterior surface of cristalline lens, and third, low counts at 419 nm and high counts at middle wavelengths. Results of a standard rod measurement program (right eye of author DN) are presented in Fig. 2B. The bleaching sequence is 3 min on, maximally 32 min off, and 5 min on. Since a measurement of such length is difficult to endure, the subject is generally allowed to lean back after 10-15 min in the dark, and to resume position about 5 minutes before light on (visible as lapses in the traces of Fig. 2B). Data were obtained at 16 deg eccentricity on the horizontal meridian, juxtapose the blind spot. The measuring field was 5.4deg, Resulting in much higher count numbers and hence, better signal to noise ratio than in the fovea1 measurements where field size was 2.4deg (note the differences in density scale between Figs 2A and B). The maximum density difference was 0.16. After completion of a measurement, a difference spectrum is available with baseline (fully bleached) defined as a linear interpolation between the mean level 60 set before light off and the last 60 set of the measurement. The dark adapted level is the mean of the last 60 set before light on. The spectra obtained in the sessions of Fig. 2 are displayed in Fig. 3. With fovea1 measuring field the spectrum peaks near 550 nm, indicative for cone pigments. For
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comparison middle (peak at 531 nm) and long (561 nm) wavelength sensitive cone absorption spectra (Baylor et al., 1987) are drawn through the data obtained (Fig. 3A). A proper comparison, which falls outside the scope of the present paper, would require an analysis that takes stray light, ratio of middle to long wavelength sensitive cones, and bleach products into account. Through the spectrum obtained at 16deg eccentricity of Dartnall nomogram with peak at 492nm is drawn (Fig. 3C). As was already noted by Rushton in 1956 the rod difference spectrum shows a substantial red shift. Also available is a curve fitting program which yields the time constants (l/e value) of regeneration for all wavelengths under the assumption that regeneration follows an exponential decay (which holds only to first approximation, cf. Smith et al., 1983; Coolen and van Norren, 1988). Data obtained with this program are plotted in Fig. 3B and D. Time constants of recovery are about 100 set with fovea1 fixation, and about 300 set at 16 deg eccentricity, corroborating the shift from cone to rod pigment. At the extremes of the spectrum low density differences prevent calculation of time constants. In conclusion, we built a retinal densitometer which is easy to use and suited for the study of kinetics and spectral characteristics of visual pigments, Fovea1 density differences were higher than ever recorded. Rcknawledgemnts-We thank C. D. van der Linden for his ~ont~bution in developing mechanical components, and G. 3. van Meel MD, J. E. E. Keunen MD and E. Bertelh MD for helpful discussions.
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