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 .
 backgroundlimbal ring ( also known as  
 circle ) contact lenses are becoming increasingly popular , especially in asian markets because of their eye - enhancing effects . 
 the pigment particles that give the eye - enhancing effects of these lenses can be found on the front or back surface of the contact lens or  
 enclosed within the lens matrix . 
 the purpose of this research was to evaluate the pigment location and surface roughness of seven types of  
 circle contact lenses.methodsscanning electron microscopic ( sem ) analysis was performed using a variable pressure hitachi s3400n instrument to discern the placement of lens pigments . 
 atomic force microscopy ( dimension icon afm from bruker nano ) was used to determine the surface roughness of the pigmented regions of the contact lenses . 
 atomic force microscopic analysis was performed in fluid phase under contact mode using a sharp nitride lever probe ( snl-10 ) with a spring constant of 0.06 n / m . 
 root mean square ( rms ) roughness values were analysed using a generalised linear mixed model with a log - normal distribution . 
 least square means and their corresponding 95% confidence intervals were estimated for each brand , location and pigment combination.resultssem cross - sectional images at 500 and 2,000 magnification showed pigment on the surface of six of the seven lens types tested . 
 the mean depth of pigment for 1-day acuvue define ( 1dad ) lenses was 8.1  m below the surface of the lens , while the remaining lens types tested had pigment particles on the front or back surface . 
 results of the atomic force microscopic analysis indicated that 1dad lenses had significantly lower root mean square roughness values in the pigmented area of the lens than the other lens types tested.conclusionssem and afm analysis revealed pigment on the surface of the lens for all types tested with the exception of 1dad . 
 further research is required to determine if the difference in pigment location influences on - eye performance . 
Methods
SEM image analysis
AFM image analysis
Results
SEM
AFM
Discussion