Standardization of Corneal Haze Measurement in Confocal Microscopy

Eye care professionals have for years assessed the clarity of the cornea by visual inspection with a slit lamp and have used a hazy stroma, epithelium, or endothelium as an indicator of a dystrophic or poorly functioning cornea. This assessment is subjective, and several devices and methods have been described to measure the brightness of light scattered or reflected from the cornea. Photomultiplier and other light-sensing systems have been mounted in the appropriate image plane of slit lamps1,2 or custom scanning optical systems3,4 to measure light backscattered from the cornea, and although many of these devices measured light from the full-thickness of the cornea, some allowed investigators to distinguish haze in the anterior, middle, and posterior cornea.5 Current interest in factors that limit vision after penetrating, anterior, or posterior (endothelial) lamellar keratoplasty have generated a need to examine vision-limiting factors such as corneal haze after these procedures and to resolve the origin of haze in the corneal depth.6–13 The haze that can develop after refractive surgery and contact lens wear has also stimulated an interest in measurement of scattered light from the corneal stroma.3,4,14–16 At best, the spatial resolution of brightness when assessed with a slit lamp and other methods has been limited to approximately a third of the corneal thickness.3,5 Spatial resolution of the source of haze was greatly improved with the use of confocal microscopes to assess backscatter from the cornea.9,17–20 With the depth of field of 11 to 26 μm,21 investigators can identify the layer associated with pathologic scatter, measured as image brightness. Identifying the layer is particularly important when scatter is suspected to increase in surgical interfaces or in the subepithelial region after procedures such as refractive surgery and lamellar keratoplasty. Most investigators have expressed corneal haze in terms of the specific units of image intensity from the instrument used for measurement. However, image brightness from the cornea or any other tissue can be compared with brightness measured at a different time in longitudinal studies or across laboratories only if the instruments are standardized so that units that express haze are equivalent. Brightness expressed as a digitized video signal is useful only for relative measurements from the same camera and varies depending on the settings of the video camera. Measurements can be standardized in two ways. First, image intensity should be expressed in terms that are meaningful and can be compared across laboratories. Second, measurements of haze must be adjusted for differences or variations in the brightness of the light source and the sensitivity of the light detector—variables that can change over hours as well as months. The standardization should be based on backscatter measured with the same instrument from a substance that does not change from day to day, can be reproduced in any laboratory, and is readily available. In this article, we propose a method of standardizing image brightness for measurement of scattered and reflected light from the cornea in confocal microscopy. We describe the scatter characteristics from the cornea from two clinical confocal microscopes with different optical designs and characteristics, the ConfoScan 4 (Nidek Technologies, Fremont, CA) and the Tandem Scanning confocal microscope (Tandem Scanning Inc., Reston, VA). This standardization technique was used to measure corneal haze from normal untreated human corneas and from a group of corneas 3 years after laser in situ keratomileusis (LASIK), to demonstrate subtle differences in haze in specific layers of the corneal stroma.