Stave J, Slowik C, Somodi S, Hahnel C, Grümmer G, Guthoff R
Augenklinik der Universität Rostock.
Klin Monbl Augenheilkd. 1998 Jul;213(1):38-44. doi: 10.1055/s-2008-1034941.
The MICROPHTHAL is a confocal slit light scanning microscope for a non-invasive in-vivo examination of corneal structures of human eyes. With this instrument even thin layers of corneal tissue can be imaged in good quality. Otherwise, blurring of single frames and deviations from the z-axis in video-sequences caused by high speed movements of the eye would normally prevent a measurement the density of keratocytes in the cornea. The goal of the investigation was optical pachymetry, the automatical measurement of the keratocytes density and a 3D-dimensional reconstruction of the central cornea in-vivo under constant imaging conditions.
We developed a low-vacuum suction cup system for stabilizing the eye in front of the microscope objective during the z-scan through the cornea. A stepmotor shifting system for the objective locates inside the suction cup with a central hole was installed underneath them icroscope. Control of this system via computer facilitated shifting the focal plane along the z-axis. The layer images were recorded using a S-VHS-tape and saved on the PC. The digital analysis was performed using a special software to automatically and off-line evaluate the density of keratocytes in combination with the 3D-reconstruction. The software also corrected the background illumination and small axial jitter. After this procedure the keratocytes density and the 3D-reconstruction in 70 images of the z-scan were calculated. We examined 47 corneas of 25 healthy probands. The range of age was 25-56 years. Independent control evaluation of the video sequences were taken manually on an INDIGO HIGH IMPACT workstation.
By assign all keratocytes to the corneal measurement volume we found a averaged density of 15,730 cells/mm3 in the central cornea. The averaged thickness of the cornea was 0.556 mm. The control valuation of identical video-sequences on the workstation accomplished the same result of 16,000 keratocytes/mm3, also similar the result of the automatically measurement with the modified software.
This modification of the microscope is a promising in-vivo tool for optical pachymetry and quantitative examination of corneal microstructures. The stabilization effect of the low-vacuum suction cup system in the front of the microscope for computer-controlled valuation of the density profile of keratocytes and the 3D-reconstruction of a central corneal volume element has produced encouraging results. Characterization of pathophysiological changes in the distribution of keratocytes after excimer laser ablation for phototherapeutic or photorefractive keratectomy, for example, can be estimated without pain for the patients.
MICROPHTHAL是一种共焦裂隙光扫描显微镜，用于对人眼角膜结构进行无创体内检查。借助该仪器，即使是很薄的角膜组织层也能获得高质量的成像。否则，由于眼睛的高速运动导致的单帧模糊和视频序列中z轴偏差，通常会妨碍对角膜中角膜细胞密度的测量。本研究的目的是在恒定成像条件下进行光学测厚、自动测量角膜细胞密度以及对中央角膜进行三维重建。
我们开发了一种低真空吸盘系统，用于在通过角膜进行z扫描时将眼睛稳定在显微镜物镜前方。在显微镜下方安装了一个用于物镜的步进电机移位系统，该系统位于带有中心孔的吸盘内部。通过计算机控制该系统，便于沿z轴移动焦平面。层图像通过S - VHS录像带记录并保存在个人电脑上。使用专门软件进行数字分析，以结合三维重建自动离线评估角膜细胞密度。该软件还校正了背景照明和小的轴向抖动。经过此过程，计算了z扫描的70张图像中的角膜细胞密度和三维重建结果。我们检查了25名健康受试者的47只角膜。年龄范围为25 - 56岁。视频序列的独立对照评估在INDIGO HIGH IMPACT工作站上手动进行。
通过将所有角膜细胞分配到角膜测量体积中，我们发现中央角膜的平均密度为15,730个细胞/mm³。角膜的平均厚度为0.556 mm。在工作站上对相同视频序列的对照评估得出相同结果，即16,000个角膜细胞/mm³，与使用改进软件的自动测量结果也相似。
这种对显微镜的改进是一种有前景的体内工具，可用于光学测厚和角膜微观结构的定量检查。低真空吸盘系统在显微镜前方的稳定作用，用于计算机控制评估角膜细胞密度分布和中央角膜体积元素的三维重建，已产生了令人鼓舞的结果。例如，在准分子激光消融用于光治疗性或光折射性角膜切削术后，无需患者痛苦即可估计角膜细胞分布的病理生理变化特征。
