Diagnostic and Surgical TechniquesQuantitative analysis of in vivo confocal microscopy images: A review
Section snippets
Image selection and analysis
When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location (central vs peripheral cornea) and depth of images. Consistency in corneal location may be maximized by the use of fixation targets--internal for slit-scanning IVCM17 or external for laser scanning IVCM.47 Accuracy in determining section depth can be maximized by using fixed landmarks (e.g. measuring keratocyte density immediately posterior to Bowman's layer) or by using devices such as
Nidek Advanced Vision Information System software
Nidek Advanced Vision Information System (NAVIS) Endothelial Analysis Software (Fig. 2), available for use with the ConfoScan IVCM software (Nidek Technologies, Fremont, CA) enables quantitative analysis of in vivo confocal images. The region of interest is easily defined in terms of area, the dimensions of which may be adjusted as required prior to analysis. For corneal endothelial images, analysis may be performed manually, automatically, or using a combination of both techniques. Manual
Corneal epithelium
The corneal epithelium typically consists of five to seven layers of cells, including superficial epithelial cells, wing cells, and basal epithelial cells. Superficial cells have most commonly been quantified in terms of cell diameter (μm) and cell density (cells/mm2); images obtained by IVCM may not be sufficient to demonstrate full detail, however.39 Mocan et al39 have objectively shown that topical fluorescein application prior to IVCM enhances the visualization of the superficial
Corneal sub-basal nerves
Corneal sub-basal nerves are easily and reproducibly imaged because of their location and orientation on Bowman’s layer. Nonetheless, the manner in which nerve density is defined has been somewhat inconsistent in the literature. The majority of studies have defined sub-basal nerve density as the total length of nerves visible within a defined area (μm/mm2 or mm/mm2),44, 48 but some investigators have only included nerve branches longer than 50 μm in their measurements.9 Others have analyzed the
Stromal nerves
Quantitative analysis of stromal nerves imaged by IVCM remains controversial. A wide range of values for stromal nerve diameter have been reported, and this variation is due to a number of factors. First, stromal nerves commonly traverse obliquely relative to the en face section of IVCM images. These nerves will therefore appear shorter than one whose path is parallel to the plane of the image (Fig. 4). The visible nerve length per frame area will also depend on the axial resolution of the
Corneal endothelium
Manual analysis of endothelial cell density simply involves counting the number of cells within a predefined frame. This traditional form of analysis only provides information regarding the cell density and number of cells counted and does not supply any morphometric data. In contrast, contemporary software applications such as the NAVIS software of the ConfoScan IVCM (see the Image Analysis Software section) provide data regarding the number of cells counted, cell density, age-matched normal
Corneal reflectivity
Measurement of corneal reflectivity is an objective method of assessing corneal haze.40 Most investigators have expressed corneal haze in terms of the specific units of image intensity from the instrument used for measurement; however, the image “brightness” of light back-scattered from corneal haze can only be compared with brightness measured at a different time in longitudinal studies or across laboratories if the IVCM instruments are standardized so that units that express haze intensity
Corneal thickness
The slit-scanning IVCM has poor repeatability for corneal thickness measurements, exhibiting the widest 95% limits of agreement both within and between sessions when compared with ultrasound, optical coherence tomography, and Orbscan.67
This is because the position of the cornea relative to the objective lens varies throughout the scan acquisition, and the error is compounded by the several second length of scanning time during which involuntary axial motion of the eye is inevitable.33
The
Immune/inflammatory cells
Zhivov et al68 were first to report in vivo evaluation of Langerhans cells within the human corneal epithelium. Quantification of Langerhans cell density is achieved by counting the number of these cells per image frame.29, 66, 68 Keratic precipitates have also been quantitatively evaluated in Fuchs heterochromic cyclitis. Characteristics such as density, diameter, area, the ratio between the total size and the body size, and number of pseudopodia are analyzed.13, 27
Reading centers
The inter-observer variability of many of the quantitative parameters discussed in this review raises the question whether there should be reading centers for IVCM image analysis. A reading center is a central facility specializing in the standard evaluation of images. Reading centers are commonly used in multicenter clinical trials. Usually, each study center requires certification by the reading center and must use the center's imaging protocol.65 In the case of IVCM, a minimum level of
Conclusion
Quantitative analysis is an increasingly common feature of studies using IVCM. It is clear that appropriate image selection and randomization are crucial prior to analysis. There are several software applications available for quantifying IVCM images, each with specific advantages and disadvantages. There are also multiple ways of defining morphological parameters and there is currently no consensus regarding “gold standard” definitions of parameters such as sub-basal nerve density, making
Method of literature search
Searches were performed using PubMed and Medline, and all years were searched. The following search terms were used: In vivo confocal microscop, AND cornea, corneal endothelium, corneal epithelium, keratocyte, sub-basal nerves, corneal haze, corneal reflectivity, corneal thickness, repeatability, and reproducibility.
All articles judged to be of relevance to quantitative analysis of IVCM images were included, and case reports were excluded. English articles and non-English language articles with
Disclosure
The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.
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