eligibility criteria included normal participants of 112 pairs of eyes ( 112 patients ) recruited from patients referred due to glaucoma suspicion from march 2008 to august 2008 at an urban tertiary - care hospital . they had normal intraocular pressure ( < 20 mmhg ) , normal findings in retinal nerve fiber layer photography , which showed no focal wedge - shaped defects or diffuse retinal nerve fiber layer loss , and normal and reliable results upon visual field testing . exclusion criteria included a best - corrected visual acuity worse than 20 / 25 in both eyes , previous medication for glaucoma , past history of optic neuropathy , previous intraocular surgery ( such as cataract removal ) , refractive error greater than -6.00 diopters , and a dysmorphic optic disc . we also excluded patients with an optic nerve head with abnormal findings , such as notching , rim thinning , excavation , presence of hemorrhage , asymmetric cup / disc ( c / d ) ratios ( > 0.2 between eyes ) , large c / d ratio ( > 0.7 ) , and noncompliance with the is nt rule . all patients had instillation of tropicamide - phenylephrine hydrochloride ( mydrin - p ; santen pharmaceutical , osaka , japan ) when the study began . optic disc photographs were then taken using a kowa nonmyd7 fundus camera ( kowa , tokyo , japan ) with a nikon d70s digital camera ( nikon , tokyo , japan ) after dilatation . optic disc images were captured using an image capture program that was presented by kowa . in the settings of the image capture program , the image format was jpeg , and the image resizing quality optic disc color analysis using imagej was done by gray scale calculation of the optic disc . three different observers calculated the gray scales at three different points for three separate times . the three points selected by each observer were as follows : 1 ) nasal rim ( nr ) , the point at three or nine o'clock from the optic disc center ; 2 ) the brightest cupping center ( bcc ) , nearly the center of cupping where the color is brightest ; 3 ) the largest inferior retinal vein ( irv ) , the point at which the inferior retinal vein comes in contact with the margin ( fig . , we applied the wand tool to the three selected points in the imagej program . this wand tool automatically creates a selection by tracing objects of uniform color or thresholded objects . the gray scale was implied to the intensity per pixel area in which it was calculated using brightness values . for color images , the histogram for gray scale was calculated by converting each rgb image into a gray scale value using the formula v = 0.299r + 0.587 g + 0.114b . we analyzed the correlation between the gray scale of the three points and the patient 's age . the intra - observer and inter - observer correlations were also evaluated to determine the reproducibility of imagej . the pearson 's correlation coefficient was calculated to evaluate the correlation between gray scales and age . an interclass correlation coefficient ( icc ) was used to evaluate intra - observer reproducibility . we calculated the pearson product - moment correlation to assess the correlation between the measurements . eligibility criteria included normal participants of 112 pairs of eyes ( 112 patients ) recruited from patients referred due to glaucoma suspicion from march 2008 to august 2008 at an urban tertiary - care hospital . they had normal intraocular pressure ( < 20 mmhg ) , normal findings in retinal nerve fiber layer photography , which showed no focal wedge - shaped defects or diffuse retinal nerve fiber layer loss , and normal and reliable results upon visual field testing . exclusion criteria included a best - corrected visual acuity worse than 20 / 25 in both eyes , previous medication for glaucoma , past history of optic neuropathy , previous intraocular surgery ( such as cataract removal ) , refractive error greater than -6.00 diopters , and a dysmorphic optic disc . we also excluded patients with an optic nerve head with abnormal findings , such as notching , rim thinning , excavation , presence of hemorrhage , asymmetric cup / disc ( c / d ) ratios ( > 0.2 between eyes ) , large c / d ratio ( > 0.7 ) , and noncompliance with the is nt rule . all patients had instillation of tropicamide - phenylephrine hydrochloride ( mydrin - p ; santen pharmaceutical , osaka , japan ) when the study began . optic disc photographs were then taken using a kowa nonmyd7 fundus camera ( kowa , tokyo , japan ) with a nikon d70s digital camera ( nikon , tokyo , japan ) after dilatation . optic disc images were captured using an image capture program that was presented by kowa . in the settings of the image capture program , the image format was jpeg , and the image resizing quality was set to medium . optic disc color analysis using imagej was done by gray scale calculation of the optic disc . three different observers calculated the gray scales at three different points for three separate times . the three points selected by each observer were as follows : 1 ) nasal rim ( nr ) , the point at three or nine o'clock from the optic disc center ; 2 ) the brightest cupping center ( bcc ) , nearly the center of cupping where the color is brightest ; 3 ) the largest inferior retinal vein ( irv ) , the point at which the inferior retinal vein comes in contact with the margin ( fig . , we applied the wand tool to the three selected points in the imagej program . this wand tool automatically creates a selection by tracing objects of uniform color or thresholded objects . the gray scale was implied to the intensity per pixel area in which it was calculated using brightness values . for color images , the histogram for gray scale was calculated by converting each rgb image into a gray scale value using the formula v = 0.299r + 0.587 g + 0.114b . we analyzed the correlation between the gray scale of the three points and the patient 's age . the intra - observer and inter - observer correlations were also evaluated to determine the reproducibility of imagej . the data were analyzed using the spss ver . 15.0 ( spss inc . , chicago , il , usa ) . the pearson 's correlation coefficient was calculated to evaluate the correlation between gray scales and age . an interclass correlation coefficient ( icc ) was used to evaluate intra - observer reproducibility . we calculated the pearson product - moment correlation to assess the correlation between the measurements . one hundred and twelve eyes of 112 patients ( 56 male and 56 female ) were enrolled in this study . the mean age of participants was 50.6 years old ( range , 11 to 82 years ) . the mean gray scale scores of the nr were 91.81  20.47 , 94.91  21.90 and 93.24  20.45 ; the bcc results were 174.84  31.63 , 179.94  31.59 and 177.76  31.56 ; and the largest irv calculations were 61.85  14.09 , 53.48  12.32 and 56.73  13.62 for observers 1 , 2 and 3 , respectively . inter - observer reproducibility for the nr , bcc and largest irv were relatively high ( icc = 0.944 , 0.860 and 0.789 , respectively ) ( table 1 ) . all observers obtained their own high reproducible values for gray scale of nr ( icc = 0.952 , 0.948 , 0.955 for observer 1 , 2 , and 3 , respectively ) , bcc ( icc = 0.932 , 0.907 , 0.947 for observer 1 , 2 , and 3 , respectively ) and largest irv ( icc = 0.923 , 0.858 , 0.873 for observer 1 , 2 and 3 , respectively ) ( table 2 ) . the order of mean gray scale score was the bcc , nr and largest irv . significant age - related differences between the values of the brightest cupping center were noted , and the gray scale score was decreased in the older participants ( r = 0.362 ) . however , the gray scale of the nr and largest irv did not show age - related differences ( r = 0.001 and 0.015 , respectively ) ( table 3 and fig . a number of previous reports have suggested a variety of methods to analyze the color of the optic disc . a scanning microdensitometric technique was introduced in 1977 that provides an objective means of measuring the degree of optic disc pallor . fundus reflectometry uses direct measurements of light from an external source that is reflected by the ocular fundus . miller and caprioli   documented that videographic image analysis using reflectometry provided rapid and quantitative measurements of optic disc pallor . even though the previous studies objectively evaluated the color of optic discs , these methods required specific measuring instruments such as microdensitometry and reflectometry . we excluded patients with a best - corrected visual acuity lower than 20 / 25 from our study because media opacities ( including corneal , cataract and vitreous opacity ) may influence the results of analyzing the color of the optic nerve head . to minimize the error of acquisition of the photography , we did not change the settings of the capture program or the digital camera during the study . we used the following three points : the nasal rim , the brightest cupping center and the largest inferior retinal vein . since the rim remnants are located mainly in the nasal disc sector in very advanced glaucoma , we thought that the color of the nasal rim would be consistent with any changes and may be suitable as an index for the standard color of the optic disc . narrowing of the retinal vessels was shown in optic neuropathy and glaucoma , and focal retinal arteriole narrowing was observed in eyes with ischemic optic neuropathy or glaucoma . however , the large inferior retinal vein is relatively stable and greater in thickness than the retinal artery , so it is easy to evaluate its color . analysis of the color of optic disc indicated that there were significant age - related differences in the gray scales of brightest cupping center ( r = 0.362 ) . however , there were no correlations between age and the gray scales of the nasal rim and the largest inferior retinal vein ( r = 0.001 and 0.015 , respectively ) . significant age - related alterations between connective tissue components were noted in the optic disc . in particular , we observed an age - related increase in elastin and collagen types iii and v , and also an age - related decrease in fibronectin . such findings may be related to the age - related changes in the brightest optic disc cupping center . varma et al .   reported that there were no age - related or refractive error related differences in the disc area or in the neural rim area and cup area ; therefore , our findings of no age - related differences in nasal rim color corresponded well with previously reported data . the highest correlation value of the nasal rim was 0.944 , and that of the brightest cupping center was 0.860 . the correlation of icv was lowest ( 0.789 ) , and this could have resulted from an observational error in point selection that may have been present . the pupil size was important to determine the intensity of the optic disc in fundus photography . if the pupil size was small , the light introduced by the flash decreased , and the color of the optic disc could be changed . to reduce the variation in the amount of light source , however , we could not exclude accidental errors when analyzing the optic disc completely . in conclusion , the gray scale of the brightest cupping center was diminished with age in the group with suspected glaucoma . imagej can be a useful objective tool to analyze the color of the optic disc and is a highly reproducible program .
purposeto evaluate the reproducibility of imagej software in analyzing the color of the optic disc.methodsone hundred twelve normal participants ( 56 males and 56 females ) were enrolled in this study . 
 the image of the optic disc was taken using kowa digital disc photo - graphy , and the gray scales of the nasal rim ( nr ) , brightest cupping center ( bcc ) and largest inferior retinal vein ( irv ) were calculated using histogram in imagej . 
 three different observers calculated the gray scales three separate times . 
 reproducibility was assessed using the interclass correlation coefficient ( icc).resultsthe mean age of the participants was 50.6 years old ( range , 11 to 82 years ) . 
 the mean gray scales of the nasal rim were 91.81 , 94.91 , and 93.24 ; those of the brightest cupping center were 174.84 , 179.94 , and 177.76 ; and those of the largest inferior retinal vein were 61.85 , 53.48 , and 56.73 for observers 1 , 2 , and 3 , respectively . 
 inter - observer reproducibility for nr , bcc and irv was considered good based upon icc values of 0.944 , 0.860 , and 0.789 for observers 1 , 2 , and 3 , respectively . 
 significant age - related differences between the values of the brightest cupping center were noted , and the gray scale score was decreased in the older participants ( p < 0.001).conclusionsthe gray scale of the brightest cupping center diminished with age . 
 imagej can be a useful objective tool with high reproducibility in the analysis of optic disc color .