Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

This method can improve the screening and selection of corneal donor tissues in eye banks, enhance the results of cornea transplantation. The main advantage of this technique is its high resolution assessment capability and that the cornea remains immersed in a closed chamber filled with storage medium throughout image acquisition, decreasing any potential risk of contamination. Demonstrating the procedure will be Maelle Vilbert, a PhD graduate from our laboratory.

To begin, position the cornea immersed in storage medium in the sample holder with the epithelium facing up. Place a clean silica cover slip provided with the sample holder on top of the cornea. Then, close the holder by gently turning its base until the sample is slightly flattened and immobilized under the cover slip, providing a relatively even imaging surface.

Take precautions to avoid any air bubbles. Apply a thick layer of ophthalmic or optical gel on the cover slip as the immersion medium. Initialize the device by turning on the power switch on the back of the device.

Illumination of a green LED on the front of the device indicates that the power is on. Ensure that the imaging stage is clear, except for the movable tray. Then click OK in the acquisition software to initialize the motors at the prompt.

To set up the device, enter a sample identifier in the designated and mandatory field. And optionally enter a sample description and study description. Next, click Acquire macro image to create a snapshot of the sample that can later be used for lateral positioning and navigation purposes.

Once satisfied, validate the image at the prompt by clicking Yes. After which the device will move the sample tray underneath the objective and perform an auto-adjustment. Ensure that the microscope objective is well immersed in the optical gel before proceeding further.

Prepare the acquisition by going to the Explore tab. Before acquiring a stack of images, navigate to the center of the cornea via the joystick or manual selection on the screen. Vary the imaging depth via rotation of the joystick, adjustment of the slider or manual keyboard input in the graphical user interface.

Then adjust the averaging value to 40 for optimal corneal imaging. Enter the corneal surface value or first image location into the depth field. Then select the Acquire tab to acquire images.

Select and set the slice spacing matching the axial resolution of the device. Enter the corneal thickness value under Number of Slices. Review parameters and acquisition time.

And when satisfied, press OK to launch the acquisition. During the acquisition, avoid contact with the table on which the microscope is positioned. To assess stromal and Bowman’s layer thickness, measure distances of corneal cross-sections.

For example, add five equally spaced points across the cross section as described in the text manuscript. Draw a line between two points of known distance according to the default field of view. Then go to the Analyze tab and select Set Scale.

Enter the known distance and unit of length in the appropriate fields. Set at 1024 pixels or 780 micrometers and click OK.Draw a line between two points of unknown distance and read the length or distance measured directly from the status bar. Record the mean and coefficient of variation.

Click on the Image tab and select Reslice Z under Stacks to determine the keratinocyte density. This will sum the stromal en face images in groups of seven to produce slices of comparable thickness. For further analysis, include all available en face slices for the anterior most stroma, 15 images for the anterior stroma, five images for the mid-stroma, and five images for the posterior stroma.

On each en-faced image, select a 300 by 300 micrometer region of interest. To enhance nucleus visualization, invert the image using Invert under the Edit tab. Adjust contrast and brightness by clicking the Image tab and navigating to Brightness-Contrast.

To manually count cell nuclei, go to Plugins and navigate to Cell Counter under the Analyze tab. Press Initialize and then select a counter type. Then count the cell nuclei by clicking on dark oval features in the inverted image, considering those landing on an image border only for two of the four sides of the image.

The selected human donor cornea was swollen after storage in organ culture medium, giving a pathophysiological model of the edematous cornea and preventing image acquisition through the entire corneal thickness due to limited penetration depth. Transfer to dextrin supplemented organ culture medium, reduced the swelling and resulted in donor corneas of normal thickness. Diseased corneas were recognized by morphological changes and typical stromal features, including a decrease in variable stromal thickness or Bowman’s layer.

Assessment of the keratinocyte density and stromal reflectivity further aided in the histology-like analysis and differentiation of normal from pathologic corneal tissues with full-field optical coherence microscopy, which is beyond the capabilities of clinical optical coherence tomography systems. After watching this video, you should have a good understanding of how to perform qualitative and quantitative histology-like analysis of the corneal stroma permitting differentiation of disease from normal human corneal tissues.