Each patient was examined on slit-lamp by two independent
ophthalmologists. The findings were presented as drawings in patient
charts or photographs. A tandem scanning confocal microscope (TSCM;
model 165A; Tandem Scanning, Reston, VA) was used for examining the
central cornea of the patients at the Alicante Institute of
Ophthalmology, Spain. The setup and operation of the confocal
microscope has been described previously.
21 23 24 Briefly,
a ×24, 0.6 NA variable working distance objective lens was used. The
field-of-view with this lens is 450 × 360 μm, and the z-axis
resolution is 9 μm. Images were detected using a Dage VE1000
low-light level camera and recorded on SVHS tape. In addition, confocal
microscopy through-focus scans (CMTF) were obtained as previously
described.
21 23 Video images of interest were digitized
using a PC-based imaging system with custom software (University of
Texas Southwestern Medical Center at Dallas) and printed using an Epson
Stylus Color 800 printer (Seiko Epson, Nagano, Japan) without image
enhancement. The central cell density of the most anterior keratocyte
layer was calculated by hand in the area of 205 × 190 μm and
reported as counts per square millimeter
(counts/mm
2). Similarly, interface particle
density was also determined. Interface particles were at most 25μ
m
2 in size and, thus, remarkably smaller than
what was regarded as keratocyte nuclei. Furthermore, interface
particles were usually brighter than keratocyte nuclei. Using the
custom software, the CMTF data were digitized onto the PC, and
intensity profile curves were calculated.
23 From each
scan, the flap thickness was measured (defined as the distance between
the surface epithelium and the flap interface characterized by
accumulation of interface particles), as well as the thickness of the
pre- and post-interface acellular zones and post-interface keratocyte
activation zone (defined as bright keratocyte nuclei and visible
keratocyte processes). A quantitative estimate of the increased
back-scattering (CMTF-haze)
21 around the flap interface
was obtained by calculating the area below the CMTF profile
corresponding to peaks originating from the structures of interest
(i.e., interface particles, increased extracellular matrix [ECM]
reflection, or activated keratocytes). CMTF-haze values were not given
for the preoperative corneas, because they did not show any extra peaks
deviating from the baseline. An average of three CMTF scans was
performed per each eye. In five eyes (one eye at 1–2 weeks, 1–2
months, and 3 months and two eyes at >6 months), an acceptable CMTF
profile was not produced because of the patients’ inability to fixate
steadily; these scans were not included in the analysis. Average values
of the measurements were used for all statistical calculations.