3. Methods All subjects are required to fill out basic information and take a survey questionnaire before and after contact lens wear. Subjects will be examined and tested before being fitted to color tinted contact lens with slit-lamp biomicroscopy and fluorescein staining (Heig Strait, Germany), intraocular pressure measurement by pneumotonometery (Topcon® CT-80), refractometer (Topcon® KR-8900), uncorrected and best corrected Snellen visual acuity (UCVA and BCVA), Schirmir test 1, tear film break-up time (TBUT), corneal topography (Bausch \& Lomb® Orb IIz), and endothelial cell density measurement by specular microscopy (Konan Noncon Robo-Sp-9000, USA). Exams are also performed at 1 week, 1, 3, and 6 months of lens wear. A special chart for all subjects is filled out during each visit During each exam, subjects will be observed for signs of conjunctival injection, conjunctival papillary hypertrophy, pannus formation, and corneal epitheliopathy. These signs are graded as shown in the table below.

Signs 0=Normal 1=Mild 2=Moderate 3=Severe Conjunctival Injection None Minimal Obvious Diffuse Papillae Size None \<0.25mm 0.25-0.5mm \>0.5mm Superficial Pannus No pannus 1 quadrant 2 quadrants \>=3 quadrants Punctate Epitheliopathy None \<1/3 of cornea 1/3-1/2 of cornea Confluent or with epithelial defect

Schirmir test 1 (basal secretion test) is performed for 5 minutes and considered abnormal if the filter strip wets \<5 mm in length. TBUT of less than 10 seconds is considered abnormal.

Impression cytology will be performed before lens wear and at 3 and 6 months post lens wear at National Taiwan University. Nitrocellulose filter paper with the pore size of 0.22 μm (Millipore GSWP 04700, Billerica, MA, U.S.A.) are cut into strips (about 4x8 mm). After a drop of topical anesthetic (proparacaine 0.5 %) application, excess tears are gently absorbed by sterile cotton swabs. Nitrocellulose strip are placed on the surface of limbal conjunctiva. After applying light pressure, the paper strips are peeled off and immediately fixed in 95% alcohol. The strips are then stained using periodic acid-Schiff (PAS ; Sigma 395132, Hattiesburg, MS, USA) and examined under light microscopy at 40X magnification and graded by mask observers following Saini et al's criteria21 as follows. Grade 1: Small round epithelial cells with a nucleus to cytoplasm ration of 1:2. Large number of deeply positive cells present. A good confluent sheet of cells is usually obtained. Grade2: A good cell sheet consisting of larger polygonal epithelial cells with a decreased nucleocytoplasmic ration of 1:3. Goblet cells reduced in number, but still deeply PAS-positive. Grade 3: Larger polygonal cells with a further decrease in nucleus to cytoplasm ratio. Reduced number of goblet cells, with reduced staining. Grade 4: Larger polygonal basophilic cells with pyknotic nuclei. Intracellular keratin often demonstrable. Absent goblet cells. Only a few loose clumps were obtainable. Goblet cell density is also graded by counting the average number of cells per 100 epithelial cells in at least four high power fields (HPF). Grade 1: \>30 goblet cells/ 4 HPF. Grade 2: 15-30 goblet cells/ 4 HPF. Grade 3: 5-15 goblet cells/ 4 HPF. Grade 4: \<5 goblet cells/ 4 HPF.

In vivo confocal microscopy imaging of the cornea will be performed before lens wear and at 3 and 6 months post lens wear by one masked examiner on all subjects at National Taiwan University. One drop of 0.5% proparacaine solution and artificial tears was instilled immediately before each exam. After asking the patients to look straight ahead, an in vivo confocal microscope (Confoscan 3.4.1; Nidek Technologies, Padova, Italy) equipped with a standard 40x water-immersion front lens captured images of the full thickness of the central cornea automatically. Each examination took approximately 1 to 3 minutes and recorded 350 images at a distance of 1.5 and 4 μm on the z-axis between successive images. During each visit, measurements were repeated with 4 um z-interval for 3 times followed by 1.5 um z-interval for 3 times. Analysis of epithelial cells morphology, epithelial thickness, and stromal reaction follows that has been published by Chen et al22-24. Corneal nerve morphology and density will also be recorded and analyzed.

Used contact lenses will be place in the provided storage cases and brought back to check for completeness of lens with no tear or chipped pieces. The lenses are then used for further material analysis by scanning electromicroscopy (SEM) and bacterial adhesion determination with personal funding. For SEM, used lenses were fixed in half strength Karnovskys fixative ( 2% paraformaldehyde and 2.5% glutaraldehyde) after gentle washing. The fixative was removed and distilled water was added for rinsing. Rapid freezing with liquid nitrogen immersion was done before being placed under vacuum overnight. The concave side of samples was mounted on aluminum stubs and sputter-coated with gold for examination under SEM (JEOL Ltd, JSM 5300, Japan) at 15kV under various magnification. For bacterial adhesion determination, used lenses are collected in sterile plates and gently dipped three times in PBS before placement in eppendorf tubes containing 1ml PBS. The lenses are then macerated and serial dilutions up to 105 are done before plating onto Muller-Hinton agar. The agar plates are incubated overnight and colony forming units are counted the next day. Number of colonies multiplied by dilution factor divided by the volume plated will give the CFU/ml/per contact lens.

All data will be entered into Excel spreadsheet (Microsoft, Inc) and analyzed using SPSS software for Windows (11.0, SPSS, Inc.). Paired t-tests will be used to compare all parameters before and after wear of the color tinted contact lenses.