the lens samples were removed from the manufacturer packaging , placed front ( convex ) surface down on a clean glass slide , cross - sectioned with a razor blade and mounted onto a lens holder for sem imaging with a hitachi model s-3400n sem at an iso 17025 accredited laboratory . the images were recorded at an acceleration voltage of 10  kv under variable pressure ( 3050 mpa ) operating mode and using a back - scattered electron detection . one 500 magnification image of each cross - section was obtained to show the entire width of the sample and the general location of the pigment particles . a 500 magnification ( and in some cases a 2,000 magnification ) image was taken in a top - down or bottom - up view . to measure the depth of the pigment particles , the uncertainty of dimensional sem measurements is about 10 per cent ( providing an estimated level of confidence of 95%).21 three to 12 lenses per lens brand were tested for each of the following seven lens types : camax color ( cc ) ; 1-day acuvue define ( 1dad ) ; one - day delight max 2 ( dm2 ) ; seed eye coffret uv ( eco ) ; freshlook one - day color ( flc ) ; naturelle daily disposable ( nat ) ; and ticon cosmetic daily ring black ( tc ) contact lenses ( table  1 ) . lens brands , manufacturers , region available , variants and powers tested listed in alphabetical order atomic force microscopic images were acquired for the seven lens types in fluid phase with a dimension icon afm from bruker nano under contact mode using sharp nitride lever probes ( snl-10 ) with a spring constant of 0.06 newton per metre . the area of lens under afm imaging and the afm tip were kept hydrated with a droplet of standard lens packing solution . within each lens brand , multiple lenses from each power tested rms roughness and peak - to - peak values were obtained by analysing three 20  m  20  m regions ( front curve , back curve , pigmented and non - pigmented ) of these images . four replicates of rms and peak - to - peak measurements were selected for each area within each lens at a given location or pigment status . thus , a total of 12 measurements were recorded for each lens at a given location or pigment area . data from rms and peak - to - peak responses were determined to be log - normally distributed . these responses were analysed separately using a generalised linear mixed model with a log - normal distribution . location ( front versus back curve ) , pigment area ( pigmented versus non - pigmented ) and lens brand were included as fixed effects . all two - way and three - way interactions between location , pigment and brand were also included as fixed effects . replicates nested within lens number and area number were modelled as random effects within each brand . least squares mean values and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination . results from the sem image analyses were used to determine the appropriate pair - wise comparisons between lens brands . pair - wise comparisons were made for each lens brand for pigment versus no pigment on the lens surfaces . in this study , since the analysis was completed on the log - scale , the afm estimates and their interval bounds were exponentiated to provide model - based estimates on the original , untransformed scale . since multiple comparisons were considered , a simulation adjustment for alpha was completed to control the the lens samples were removed from the manufacturer packaging , placed front ( convex ) surface down on a clean glass slide , cross - sectioned with a razor blade and mounted onto a lens holder for sem imaging with a hitachi model s-3400n sem at an iso 17025 accredited laboratory . the images were recorded at an acceleration voltage of 10  kv under variable pressure ( 3050 mpa ) operating mode and using a back - scattered electron detection . one 500 magnification image of each cross - section was obtained to show the entire width of the sample and the general location of the pigment particles . a 500 magnification ( and in some cases a 2,000 magnification ) image was taken in a top - down or bottom - up view . to measure the depth of the pigment particles , the uncertainty of dimensional sem measurements is about 10 per cent ( providing an estimated level of confidence of 95%).21 three to 12 lenses per lens brand were tested for each of the following seven lens types : camax color ( cc ) ; 1-day acuvue define ( 1dad ) ; one - day delight max 2 ( dm2 ) ; seed eye coffret uv ( eco ) ; freshlook one - day color ( flc ) ; naturelle daily disposable ( nat ) ; and ticon cosmetic daily ring black ( tc ) contact lenses ( table  1 ) . lens brands , manufacturers , region available , variants and powers tested listed in alphabetical order atomic force microscopic images were acquired for the seven lens types in fluid phase with a dimension icon afm from bruker nano under contact mode using sharp nitride lever probes ( snl-10 ) with a spring constant of 0.06 newton per metre . the area of lens under afm imaging and the afm tip were kept hydrated with a droplet of standard lens packing solution . within each lens brand , rms roughness and peak - to - peak values were obtained by analysing three 20  m  20  m regions ( front curve , back curve , pigmented and non - pigmented ) of these images . four replicates of rms and peak - to - peak measurements were selected for each area within each lens at a given location or pigment status . thus , a total of 12 measurements were recorded for each lens at a given location or pigment area . data from rms and peak - to - peak responses were determined to be log - normally distributed . these responses were analysed separately using a generalised linear mixed model with a log - normal distribution . location ( front versus back curve ) , pigment area ( pigmented versus non - pigmented ) and lens brand were included as fixed effects . all two - way and three - way interactions between location , pigment and brand were also included as fixed effects . replicates nested within lens number and area number were modelled as random effects within each brand . least squares mean values and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination . results from the sem image analyses were used to determine the appropriate pair - wise comparisons between lens brands . pair - wise comparisons were made for each lens brand for pigment versus no pigment on the lens surfaces . in this study , since the analysis was completed on the log - scale , the afm estimates and their interval bounds were exponentiated to provide model - based estimates on the original , untransformed scale . since multiple comparisons were considered , a simulation adjustment for alpha was completed to control the cc , dm2 , eco and tc lenses were found to have pigments on the back surface ( concave side ) of the contact lens , while the pigments for nat and flc were found on the front surface ( convex side ) of the contact lens . the pigment particles were visible on the lens surface in top - down / bottom - up views , as well as in cross - sectional view appearing to extend from an average depth of 1.3 m to 3.5 m up to and including the lens surface for a depth range of 0.0 to 4.3m . the pigment particles for the 1dad were buried below the front surface of the lens at an average depth of 8.1 m with a range of 7.6 to 9.1 m ( table  2 ) . depth range for pigments from the lens surface when imaged by scanning electron microscope ( sem ) for each lens type 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable figures 1 
 7 show the cross - sectional depths at 2,000 magnification for each of the lens types . the images show no pigment particles on the top of the 1dad lenses , while a particle layer is on the surface of all other lenses tested . these results were used to determine the statistical comparisons to be completed for the afm findings ( surface pigmented lenses versus non - surface pigmented lenses ) . cross - sectional view at 2,000 magnification showing pigment enclosed within the lens matrix ( lm ) below the front lens surface ( a ) and an image of the lens front surface at 500 magnification showing no pigment particles ( b ) . 1dad : 1-day acuvue define . scanning electron microscopic ( sem ) images of cc . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . flc : freshlook one - day color . scanning electron microscopic ( sem ) images of nat . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . the results of the afm analysis indicated that 1dad lenses had a statistically significantly lower rms value in the pigmented area than cc , dm2 , eco , tc , flc and nat contact lenses . table  3 displays the measures for each lens type by curve / print aspect combinations . table  4 and figure  8 summarise the statistical findings and graphically show the mean roughness values for each lens type in the different lens areas . figure  9 shows representative afm images of the pigmented areas for each lens type . atomic force microscopic average and range  sd ( and range ) for root mean square ( rms ) and  peak - to - peak values for the front and back surfaces , pigmented and non - pigmented areas 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . average root mean square ( rms ) roughness values determined by atomic force microscopy on the non - pigmented area of the front lens surface ( non - pig fs ) and back lens surface ( non - pig bs ) as well as the pigmented area of the front ( pig fs ) and back lens surfaces ( pig bs ) . pigment location was determined by scanning electron microscopy ( np : no pigment on the lens surface , bs : back surface pigment , fs : front surface pigment ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . atomic force microscopic images of pigmented areas for 1dad ( a ) , tc ( b ) , cc ( c ) , dm2 ( d ) , eco ( e ) , nat ( f ) , and flc ( g ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . statistical analysis of atomic force microscopy root mean square ( rms ) values for lenses found to have surface pigment versus the lens with pigments enclosed within the lens matrix 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . p - values are all versus 1dad additionally , 1dad lenses had roughness values that were more consistent between the pigmented and non - pigmented areas compared to the other lens types measured . rms roughness values for 1dad ranged from three to 18  nm , while rms values for the other lens tested ranged from two to 188  nm . cc , dm2 , eco and tc lenses were found to have pigments on the back surface ( concave side ) of the contact lens , while the pigments for nat and flc were found on the front surface ( convex side ) of the contact lens . the pigment particles were visible on the lens surface in top - down / bottom - up views , as well as in cross - sectional view appearing to extend from an average depth of 1.3 m to 3.5 m up to and including the lens surface for a depth range of 0.0 to 4.3m . the pigment particles for the 1dad were buried below the front surface of the lens at an average depth of 8.1 m with a range of 7.6 to 9.1 m ( table  2 ) . depth range for pigments from the lens surface when imaged by scanning electron microscope ( sem ) for each lens type 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable figures 1 
 7 show the cross - sectional depths at 2,000 magnification for each of the lens types . the images show no pigment particles on the top of the 1dad lenses , while a particle layer is on the surface of all other lenses tested . these results were used to determine the statistical comparisons to be completed for the afm findings ( surface pigmented lenses versus non - surface pigmented lenses ) . cross - sectional view at 2,000 magnification showing pigment enclosed within the lens matrix ( lm ) below the front lens surface ( a ) and an image of the lens front surface at 500 magnification showing no pigment particles ( b ) . 1dad : 1-day acuvue define . scanning electron microscopic ( sem ) images of cc . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the back surface ( bs ) of the lens ( a ) and an image of the lens back surface at 2,000 magnification showing visible pigment particles ( b ) . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . flc : freshlook one - day color . scanning electron microscopic ( sem ) images of nat . cross - sectional view at 2,000 magnification showing pigment on the front surface ( fs ) of the lens ( a ) and an image of the lens front surface at 2,000 magnification showing visible pigment particles ( b ) . the results of the afm analysis indicated that 1dad lenses had a statistically significantly lower rms value in the pigmented area than cc , dm2 , eco , tc , flc and nat contact lenses . table  3 displays the measures for each lens type by curve / print aspect combinations . table  4 and figure  8 summarise the statistical findings and graphically show the mean roughness values for each lens type in the different lens areas . figure  9 shows representative afm images of the pigmented areas for each lens type . atomic force microscopic average and range  sd ( and range ) for root mean square ( rms ) and  peak - to - peak values for the front and back surfaces , pigmented and non - pigmented areas 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . average root mean square ( rms ) roughness values determined by atomic force microscopy on the non - pigmented area of the front lens surface ( non - pig fs ) and back lens surface ( non - pig bs ) as well as the pigmented area of the front ( pig fs ) and back lens surfaces ( pig bs ) . pigment location was determined by scanning electron microscopy ( np : no pigment on the lens surface , bs : back surface pigment , fs : front surface pigment ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . atomic force microscopic images of pigmented areas for 1dad ( a ) , tc ( b ) , cc ( c ) , dm2 ( d ) , eco ( e ) , nat ( f ) , and flc ( g ) . 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . statistical analysis of atomic force microscopy root mean square ( rms ) values for lenses found to have surface pigment versus the lens with pigments enclosed within the lens matrix 1dad : 1-day acuvue define , cc : camax color , dm2 : one - day delight max 2 , eco : eye coffret , tc : ticon cosmetic ring black , flc : freshlook one - day color , nat : naturelle daily disposable . p - values are all versus 1dad additionally , 1dad lenses had roughness values that were more consistent between the pigmented and non - pigmented areas compared to the other lens types measured . rms roughness values for 1dad ranged from three to 18  nm , while rms values for the other lens tested ranged from two to 188  nm . the sem imaging results showed , both at the lens surface and throughout the depth of the lens , that the limbal ring pigment used for 1dad was enclosed within the lens matrix , and therefore would not be in direct contact with the ocular surface when worn . for the other lens types evaluated , each lens was found to have pigment at the surface level , some on the front surface and some on the back . the differences in pigment location are likely to be due to differences in the manufacturing processes . the testing methodology used for this analysis with afm measured the samples from similar locations , which resulted in a consistent comparison among the lenses tested . afm analysis showed 1dad lenses had lower roughness values ( that is , a smoother surface ) than the other limbal ring lenses tested within the pigmented area of the lens . additionally , 1dad had a more consistent roughness profile regardless of the curvature or pigmented side than any of the other limbal ring contact lenses tested . differences in surface roughness between the pigmented and non - pigmented areas are likely the result of pigment location ( due to the fact that this can be visualised at a low magnification ) but surface roughness of the non - pigmented areas can be related to manufacturing methods as well as material properties of the bulk material.22,23 although there are no thresholds of acceptable or non - acceptable rms roughness for contact lenses , values in the range of one to 11 nm for cast - moulded contact lenses can be considered normal and smooth . biomaterials research evaluating the effect of surface roughness and surface wettability on bacterial attachment to glass has suggested even nanoscale levels of roughness can impact bacterial adherence . mitik - dineva and colleagues23 found changes in surface wettability had no impact on bacterial attachment but the roughness of the material was found to play a role . although mitik - dineva and colleagues conducted their research using glass plates , later work published by giraldez and yebra - pimentel11 would suggest surface roughness is also a factor for bacterial adhesion with contact lenses . additional research is needed to determine to what effect pigment on the surface of a contact lens may have on product performance , including the adhesion of bacteria and the levels of bacteria that may be considered to be clinically relevant . it is important for eye - care providers to understand potential differences between limbal ring contact lenses , when determining what to prescribe for their patients , beyond the consumer - desired eye - enhancement effects . with the growing market and consumer interest in limbal ring lenses , pigment location and surface roughness although there has been some recent research reporting potential clinical implications with limbal ring contact lenses that have pigment on the surface of the lens , the clinical effects of pigment location of these lenses needs to be evaluated further .