Possible Errors in the Measurement of Retinal Lesions

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1 Possible Errors in the Measurement of Retinal Lesions / V. Arnold*]. W. C. Gates j and K. M. Taylor* Purpose. To determine the accuracy of the measurement of structure in the retina from fundus photographs. Method. A model eye was developed to measure the retinal image size of the Zeiss fundus camera. A X10 microscope objective with a focal length of mm was used together with a microscope graticule. Results. Under emmetropic conditions the difference between the calculated and the measured magnification was 8.6%. The total change in magnification from myopia to hyperopia was 24.63% to +18.1%. When the camera position was altered with respect to the model eye by ±5 mm under myopic conditions the change in magnification was 4.5% and +6.8%. In the hyperopic condition the change was 5.6% to +4.6%. Conclusions. Measuring the size of structure in the retina from photographs may therefore prove to be inaccurate but in some circumstances determining the percentage change in size may prove more reliable. Invest Ophthalmol Vis Sci. 1993;34: ijince the introduction of the fundus camera, many attempts have been made to determine the size of various structures in the retina. Photogrammetric methods have also been employed with the fundus camera to assess the depth of the optic cup. To determine these parameters it is essential to know the exact magnification of the camera image. Of particular interest to our cardiac surgery unit is to determine the level of capillary dropout in fluorescein angiograms obtained during cardiopulmonary bypass surgery. 1 " 3 In these studies, using a method of intraoperative fluorescein angiography, and the subsequent use of digital image analysis, both developed by our group, 47 we have shown that capillary dropout is present in the retina in a large proportion of patients undergoing cardiopulmonary bypass surgery. Because it is now possible to not only determine those vessels that re- From *Cardiac Surgery Unit, Royal Postgraduate Medical School and the fdepartment of Surveying and Photogrammetry, University College, London, United Kingdom. Supported in part by the British Heart Foundation. Submitted for publication: August 14, 1992; accepted January 26, Proprietary interest catagory: N. Reprint requests:]. V. Arnold, Cardiac Surgery Unit, Royal Postgraduate Medical School, Du Cane Road, London, WJ2 ONN, England. main unfilled throughout the entire sequence automatically, but also to ascertain the time for the delayed filling of some of the vessels, 8 it would seem that a method of determining the exact length of the vessels, enabling a direct comparison between large numbers of patients is desirable. The same method would also allow the determination of other parameters such as vessel caliber and areas of lesions. Many attempts have been made to determine the exact magnification of the retinal image including those with ametropic Q-l 9 errors. The magnification of the the image from the Zeiss (Welwyn, Garden City) fundus camera is X2.43 for emmetropic eyes and varies from XI.79 with a refractive error of 16 diopters to X3.1 at +16 diopters (personal communication, Carl Zeiss, Oberkochen, July 1990). These figures agree quite closely with those reported by Bengtsson and Krakau. 10 The same figures are obtained if the formula specified by Littman is used. 13 Littman states that the magnification is equal to the focal length of the fundus camera divided by the focal length of the eye. This formula can only be expected to apply to emmetropic eyes of varying length and since most refractive errors are axial, the specified 2576 Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8 Copyright Association for Research in Vision and Ophthalmology

$Retinal Measurement Errors 2577 magnification with different refractive errors must be considered suspect.$

2 Retinal Measurement Errors 2577 magnification with different refractive errors must be considered suspect. To verify this, we devised a "model eye" based on a X10 microscope objective, having a focal length close to that of Gullstrands model eye and used this to determine changes in magnification that occur under different "ametropic" conditions and changes in camera position with respect to the "model eye." In a study (unpublished data) determining the effect of different pharmacologic interventions during cardiopulmonary bypass, we subjected 26 dogs to retinal fluorescein angiography. At the end of the experiment, the dogs were killed for histologic studies and the study eye was enucleated for the assessment of the magnification of the photographic image. MATERIALS AND METHODS The animals were treated according to the ARVO recommendations on the use of animals in research. We used the "magnification" method to measure the focal lengths of both the fundus camera and the microscope objective. A scale is placed in front of the lens to be tested and a sharp image formed, and the distance between some point on the lens and the image is measured (V) together with the image size (M). The object is then moved by a distance and a sharp image formed and the same parameters are measured again. The focal length of the lens is then the difference between the two image distances divided by the difference in image magnification sizes (dv/dm). To measure the change in magnification with refractive error and variation in camera position a flat scale divided into 0.1-mm intervals was mounted on a microscope stage and the microscope objective was clamped in position directly above it. The fundus camera was then positioned vertically relative to the objective. Water, having a similar refractive index to the media in the eye (1.336) was introduced between the objective and the scale. The fundus camera draw tube was then set to the emmetropic position (infinity). This was determined with a collimator, which provides an image of a target at infinity. The scale was transilluminated with an electronic flash synchronized with the camera. The microscope stage was then adjusted to provide a sharp image of the scale in the center of the field. Ideally, this scale should be curved because the optics of the fundus camera are designed for use with a curved field so that a flat scale only approximates to the retina in practice, but since only the central 0.5 mm of the scale was used for measurement, the error introduced is considered to be very small. Photographs were obtained of the scale at different positions of the draw tube from minimum to maximum extension and the change in distance of the scale relative to the objective was recorded. The draw tube of the fundus camera was then set to minimum extension (myopic condition) and photographs were obtained when the camera position was altered by ± 5 mm. It was necessary to change the position of the scale slightly to achieve sharp focus. This was repeated with the camera tube at maximum extension (hyperopic condition). The photographs were recorded on Kodak Technical Pan film developed in Kodak Dl9 developer (Eastman Kodak, Rochester, NY) for 4 min at 20 C. After processing, the negatives were placed in a Zeiss (Jena) microfilm reader and projected at a magnification of XI 4.8 on the screen. The central 0.5 mm of the scale was used to determine the magnification at the image plane of the fundus camera. To calculate the image magnification of the dog retina the optic disk was photographed with the fundus camera using red-free illumination. Two photographs were taken, one with a contact lens in place and one without in 11 of the 26 dogs. The contact lens was used throughout the study with saline to maintain clarity. The position of the draw tube on the camera was measured and it was found that the position altered when the contact lens was removed. This meant that the contact lens and saline introduced a refractive error. According to Littman, 13 this should not produce a change in image size because the error is refractive rather than axial. After enucleation, the external axial length was measured and the eye was dissected and the disk photographed using a Zeiss dissecting microscope with camera attachment. The magnification at the film plane with this instrument was X9.1. Because the disks are of an irregular shape, the area rather than the width was measured and compared with the area of the image obtained with the fundus camera. To determine these areas we used a Contextvision image analysis system (Systems AB Linkopung, Sweden) to trace the areas on the computer monitor using a mouse-controlled cursor. RESULTS The focal length of the microscope objective was found to be mm, which differs from that of Gullstrands eye by 3.9%. The focal length of the fundus camera was mm. According to Littman's formula, this should provide a magnification of X2.54 with the model eye and X2.44. with gullstrands eye. In practice, we found that the actual magnification with the model eye was X2.76, a difference of 8.6% from that calculated. The change in magnification for the different ametropic conditions is shown in Figure 1. The minimum magnification was X2.08 and the maximum X3.26, the change in magnification therefore is % for hyperopia and 24.63% for myopia. The change in the position of the scale was mm over

3 2578 Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8 Calculated verses measured magnification using model eye Magnification with and without contact lens Calculated Camera extension (mm) FIGURE l. The difference between the calculated and the measured magnification. the complete range of the camera draw tube extension, representing an increase of 8.33 mm from the emmetropic to myopic position and a decrease of 3.69 mm for the hyperopic position. When the camera was set to the minimum extension of the draw tube, (myopia) and the whole camera moved ±5 mm with respect to the model eye, the change in magnification was; 5 mm, XI.99, and +5 mm, X2.23 When the camera was focused for hyperopia, that is, maximum extension, the change in magnification was from X3.08 when too far away and X3.41 when too close (Figure 2). In practice, this degree of error should not arise if the operator is careful when positioning the camera but it serves to demonstrate the possible error caused by incorrect camera positioning when large refractive errors are present. In the canine eye, The mean magnification was 3.89 with the contact lens and 4.08 without, a mean Change in magnification with variation of distance between camera and model eye Camera position 80 Myopic condition Hyperopic condition FIGURE 2. The change in magnification in the presence of a high refractive error when the camera position is adjusted by ± 5 mm With contact lens FIGURE 3. The change in magnification with and without a contact lens. difference of 4.8% (Figure 3) This is in contradiction to Littman 13 who states that the image size does not change when the error is caused by the refractive component. DISCUSSION Pach et al 14 have previously investigated the changes in magnification with refractive error, camera decentration, and by varying the distance between the camera and the eye. Their results are somewhat confusing. They determined that there was no change in magnification when the camera distance was altered by 1.5 cm and +15 cm. This is hardly surprising because their subject was emmetropic and therefore the fundus is at infinity. They also found a change in magnification by decentering the image when the Zeiss fundus camera was used. They report an increase in image size of 20% when the disc was at the edge of the field, in two emmetropic subjects. We randomly selected color photographs of ten eyes taken with the Zeiss fundus camera. The disk area is sometimes difficult to define, so the same structure on the disk was measured, at the center of the field, and at the edge. The refractive condition of the subjects were unknown. We found a mean increase in image size of 5.5% (range %; Fig. 4). This represents a high degree of error but not as high as that in Pach's report. Our findings in the case of refractive ametropia also differ from their results. We have shown that several factors influence the determination of the magnification, and therefore the size of various parameters within the fundus photograph. Several methods have been proposed with 4.6

Retinal Measurement Errors 2579 % change 10-, 8-6- 4-2- Percentage change in magnification at edge of field + + + FIGURE 4. The change in magnification relative to position within the field.

4 Retinal Measurement Errors 2579 % change 10-, Percentage change in magnification at edge of field FIGURE 4. The change in magnification relative to position within the field. which the magnification may be calculated. The most common approach seems to be to obtain values of the axial length, corneal curvature, lens thickness, and the depth of the anterior chamber. However, Snead et al 15 have reported errors in ultrasound measurements for axial length when hard and soft probes are used. Even if the calculations were accurate, there could still be errors introduced by incorrect camera position when high refractive errors are present. Bengtsson et al 10 calculated the change in magnification with refractive error, based on Gullstrand's model eye. We have shown that the method is not accurate, partly because when the calculation is based on the first focal length of the eye, the error is in fact a change in the axial length but the eye still remains emmetropic, whereas most refractive errors are axial. Our results show that the actual magnification is higher than that calculated for emmetropic eyes. Baumbach et al 9 have reported a method of "Absolute ocular fundus dimensions" using an interference pattern generated by a laser coupled to the fundus camera. The fringes appear to be derived from coherent illumination of a regular series of apertures, however, a transverse equally spaced series of coherent point sources (eg, derived from a laser) would produce interference in the form of a family of confocal hyperboloids of revolution. The pattern produced on a flat screen would have only the central fringe straight. Outer fringes would be increasingly curved outward, with increasing spacing. The series of secondary sources is produced by multiple reflections in a thin transparent plate coated with highreflection films. The plate appears to be canted at about 20 degrees to produce the stated transverse separation of the secondary sources, and must therefore been about 0.25 mm thick. The laser was focused down on this component, and the original set of secondary sources would have appeared to be formed behind the primary focused "source," as a consequence of the successive path increments of each double transit of the space between the reflecting surfaces. Consequently, the secondary sources are not only transversely separated by 31 /zm, but are separated axially also, by 4/3 times the thickness of the plate, or approximately 300 /im. The effect of such an oblique but mainly axial separation of sources would be to produce concentric interference rings of varying spacing. Because the plate is tilted at such a large angle, the pattern observed is only a small part of the outer field and appears as nearly equidistant curved fringes. This seems to be the case in the photographs shown. Furthermore, the observed pattern through an aperture close to the secondary source means that the form of the pattern observed will appear to vary very little with the distance and form of the surface reflecting the pattern, and is therefore of no use for locating or studying the height or form of local features in the surface. Most importantly, the magnification between the tilted plate and the retina is not considered: the many refractions and changes of media make this most complicated. Furthermore, the calculations used in this study are based on measurements made on, or very close to the optical axis. It cannot be assumed that the magnification at the edge of the field is the same at the periphery. CONCLUSIONS We have developed a model eye to determine the changes in image size under different degrees of ametropia and also when the camera position is altered. The results show that the measured magnification differs significantly from the calculated data when previously described formulas are used. Our results are based on measurements made close to the optical axis. Under varying degrees of ametropia, not only the length of the eye will change but also the amount of curvature and in these circumstances the area photographed may no longer form part of a perfect sphere; therefore, the image size, either measured or calculated on the optical axis, will not apply to the parts of the image at the edge of the field. This is will add significantly to the error. We conclude that most current methods for determining the size of structure in the fundus photograph suffer from significant errors. This will be particularly relevant when attempting to measure blood velocity using the laser Doppler technique in which it is important to calculate the cross-sectional area of a vessel to determine the volume flow. One possible solution to this problem is not to depend on the assumption that the magnification is

2580 Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8 uniform but to compare differential changes of the structures in the same part of successive fields.

5 2580 Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8 uniform but to compare differential changes of the structures in the same part of successive fields. The use of digital image analysis can help to overcome some of these problems. We have previously reported a method of digitizing fluorescein angiograms in which the detail in two frames obtained on different occasions can be spatially and geometrically registered. 4 ' 5 This process automatically corrects for magnification errors and therefore the two fields selected for analysis will be identical in size regardless of the actual magnification. On this basis, percentage differences can be calculated and therefore a comparison between patients is possible. Work is currently in progress by our group to automatically identify vessels of different sizes to enable differentiation between larger vessels and capillaries. If digital image analysis is applied to retinal photographs and automatic spatial and geometric alignment is achieved, the calculation of the magnification may be alleviated in many cases. This will probably not apply in cases of stereophotogrammetry for which accurate depth measurements are required. Further research is required before reliable measurements can be made from retinal photographs. Key Words retinal photographs, magnification error, measurement, image analysis References 1. Blauth C, Arnold J, Kohner EM, Taylor KM. Retinal microembolism during cardiopulmonary bypass demonstrated by fluorescein angiography. Lancet. 1986;ii: Blauth C, Arnold JV, Schulenburg WE, McCartney A, Taylor KM. Cerebral microembolism during cardiopulmonary bypass: retinal microvascular studies in vivo using fluorescein angiography. J Thorac CardiovascSurg. 1988;95: Blauth CI, Smith PLC, Arnold JV, Jagoe JR, Wootton R, Taylor KM. Influence of oxygenator type on the prevelence and extent of microembolic retinal ischaemia during cardiopulmonary bypass: assessment by digital image analysis. J Thorac Cardiovasc Surg. 1990;99: Jagoe JR, Blauth CI, Smith PL, et al. Quantification o_ retinal damage during cardiopulmonary bypass: comparison of computer and human assessment. Proc IEE. 1990; 137 Part 1(3):17O Jagoe J, Blauth C, Smith P, Arnold JV, Taylor KM, Wootton R. Automatic geometrical registration of fluorescein retinal angiograms. Computers and Biomedical Research. 1990; 23: Arnold J, Jagoe R, Blauth C, Wootton R, Smith PLC, Taylor KM. Computerised image analysis for the detection of capillary dropout in fluorescein angiograms. Invest Ophthal Vis Sci. 1990;31(suppl): Arnold JV, Blauth CI, Smith PLC, Jagoe JR, Wootton R, Taylor KM. Demonstration of cerebral microemboli occurring during coronary artery bypass graft surgery usingfluorescein angiography./ Audiov Media Med. 1990;13(3): Arnold JV, Jagoe R, Blauth C. The detection of microembolic lesions from the complete angiographic sequence obtained during cardiopulmonary bypass using digital image analysis. Invest Ophthalmol Vis Sci. 1991;32: Baumbach P, Rassow B, Wesemann W. Absolute ocular fundus dimensions measured by multiple beam interference fringes. Invest Ophthalmol Vis Sci. 1989;30: Bengtsson B, Krakau C. Some essential features of the Zeiss fundus camera. Ada Opthalmol. 1977; 55: Littmann H. Zur bestimmung der wahren grosse eines objektes auf dem hintergrund des lebenden auges. Klin Monatbl Augenheilkd. 1982; 180: Lotmar W. Dependence of magnification upon the camera to eye distance in the Zeiss fundus camera. Ada Ophthalmol. 1984;62: Littman G, Summerer G. Fundus photography on Polaroid film. Zeiss Inf. 1970; 76: Pach J, Pennel DO, Romano PE. Optic disc photogrammetry: magnification factors for eye position, centration, and ametropias, refractive and axial; and their application in the diagnosis of optic nerve hyperplasia. Ann Ophthalmol. 1989;21: Snead MP, Rubinstein MP, Hardman LS, Havvorth SM. Calculated verses A-scan result for axial length using different types of ultrasound probe tip. Eye. 1990;4:7l8-722.

Real-Time Blood Velocity Measurements in Human Retinal Vein Using the Laser Speckle Phenomenon

Real-Time Blood Velocity Measurements in Human Retinal Vein Using the Laser Speckle Phenomenon Miyuki Nagahara,* Yasuhiro Tamaki, Makoto Araie* and Hitoshi Fujii *Department of Ophthalmology, University