角膜共焦点顕微鏡：末梢神経障害で小型ファイバ病理を定量化するための新しい非侵襲的方法

This video demonstrates a procedure to image corneal C fibers using the non-invasive technique of corneal confocal microscopy or CCM enabling quantification of nerve fiber damage. First, the focal plane of the lens on the CCM is adjusted and visco tear gel is applied to the lens before fitting the disposable cap. Anesthetic drops and visco tear gel are then applied to the eye.

Next, the camera is adjusted to make contact between the gel on the front of the lens and the patient's cornea. Finally, the depth of the focus plane is adjusted and images of corneal nerves from the central part of Bowman's layer are captured. Morphological changes in corneal nerve fibers can be observed at quantify neuropathy.

This is a very novel technique which has actually previously been used in the world of optometry and ophthalmology, but over the past eight years or so, we have used it very effectively to study and assess diabetic neuropathy and other peripheral neuropathies. And the main advantage of this technique is that it allows you to quantify small fiber damage at a very early stage, and then s stratify the severity of neuropathy in the patients entirely non-invasively, and we can do that very rapidly with a two minute scan, which can then be quantified in terms of severity of neuropathy and therefore this we hope supersedes some of the other much more invasive techniques such as skin biopsy and nerve biopsy. Whilst it looks overall a simple technique, actually, it requires definite steps by which you capture the image optimally and then you quantify the image.

And really we want to make that whole process clear and apparent to anybody else who may wish to use this technique. The initial step in examination using the HRT corneal confocal microscope is preparation of the objective lens tip at the objective tube of the laser scanning camera. Set the refraction to plus 12 diopters, and then adjust the camera to the lowest position and lock the lens by rotating it anti-clockwise.

Apply a large homogenous bubble free P size drop of visco tears onto the lens tip. Remove a tomo cap from its sterile container and mount it over the lens Tip as far as possible over the holder, such that the gel forms a meniscus between the objective lens and the cap. Be careful not to touch the front surface of the tomo cap during mounting.

Now, move the laser scanning camera as far backwards as possible on the camera mount the inner and outer surface of the tomo cap. Both appear as a bright laser reflection on the display monitor. Adjust the focal plane adjustment wheel until a bright reflection is observed, indicating that the lens is focused within the front of the cap.

The depth value shown in the focus position display should now be between minus 150 micrometers and plus 150 micrometers. Reset the depth setting to zero. Now let's see how to prepare the patient.

After anesthetizing the subject's eyes with the drop of 0.4%ate hydrochloride, lubricate the front of the eye with visco tears. See the subject comfortably with their chin on the chin rest, ensuring that their forehead is pressed firmly against the forehead bar. Examination is made easier by instructing the patient to fixate on the outer fixation light.

With the eye not being examined, the compact size of the microscope head ensures that the subject's contralateral eye is not obscured. Enabling the use of distance targets. Position the CCD camera such that its optical access runs perpendicular to the optical access of the laser scanning camera.

The camera should be on the patient's right side when imaging the right eye and vice versa. At this position, the front surface of the tomo cap is seen in the center of the CCD camera. Live image.

Move the laser scanning camera towards the patient until the patient's cornea is at a distance of about five to 10 millimeters from the tomo cap. Then use the vertical and horizontal access adjustment knobs on the camera mount to move the laser scanning camera up and down and left and right until the tomo cap is positioned in the center of the patient's cornea. Now ask the patient to open his or her eyes wide as possible.

Slowly move the laser scanning camera toward the patient until the tomo cap contacts the patient's cornea. At this stage, check that the red laser light is in the center of the cornea and that the reflection of the laser beam from the cornea occurs exactly at the anterior pole of the cornea. There is minimal contact between the lens and the cornea with optimal adjustment.

A thin gel bridge between the tomo cap and the cornea is visible on the CCD camera live image. If there is too much pressure between the tomo cap and cornea, then there is a flattened appearance in the cornea via the CCD camera. Live image and strayer appear on the CCM image.

After adjusting the patient turn on the HR three laser camera, the image acquisition window will appear on the screen. Use the CCD image to position the tomo cap and laser light on the patient's cornea. The CCM has three options for capturing images.

The sequence scan allows up to 100 images to be acquired with an adjustable frame rate. A frame rate between 30 and one frames per second is selected. And based on this, a movie can be acquired with a duration of three to 100 seconds.

In the volume scan mode, the camera automatically acquires a series of 40 images at consecutive focal planes. A total depth of 85 micrometers can be scanned, and the focal distance between two consecutive images is approximately 2.1 micrometers. The last option is section mode, which will be utilized for this experiment.

This mode enables optimal imaging with clear single images acquired and stored each time the foot switch is pressed. This mode is used for capturing images from the whole corneal layer, including Bowman's layer in the center of the cornea. After capturing sufficient numbers of images, other modes can be used including sequence and volume.

After a few scans, the focal plane of the lens can be changed to measure the depth of a corneal cell layer relative to the corneal surface by bringing the epithelial cell layer into focus and then clicking the reset button to set the depth value to zero. After the examination is complete, turn the camera off by clicking the camera power button in the acquisition window. Make the patient aware that he or she should not rub his or her eyes until the local anesthesia has worn off.

Remove the tomo cap from the camera, dispose of it, and then clean the microscope lens with a cotton swab moistened with distilled water. Now let's see how to analyze an image and quantify nerve fiber parameters to review and analyze existing examinations. First, select the appropriate patient record in the database window.

The image viewing window provides an overview of existing cornea examinations for the selected patient Examinations performed on different days are stored in separate visit tabs. The three different scan types are represented by three different icons. In the HRT three cornea visit tab of the image viewing window of the Heidelberg Eye Explorer.

To survey the cornea image, double click the appropriate image icon and then press show now to export the appropriate images for further analysis, click export on the toolbar and then select the folder to save the images to. Because the main focus of this research is on Bowman's layer and the corneal nerves only the images from this layer will be analyzed. Five to six frames from Bowman's layer are selected for each subject at different depths of the cornea.

Image analysis is conducted with specialized software developed by our group called CCM Image Analysis Tools, version 0.64. Main parameters for corneal nerves are measured with the software nerve fiber density, which is defined as the number of main nerves per frame. Nerve branch density is the total number of main branches per frame.

Nerve fiber length is the total length of main nerves and main branches per frame nerve fiber. Tortuosity is the tortuosity of the main nerves per frame. To open a CCM image, go to the file and then open.

Once the image is loaded, its size and type are displayed in the image information box on the top left hand side of the control panel, the default scale is displayed in the edit line of the image information. If the scale is different, modify the number in the edit line. To start analyzing the image, select the parameter you want to measure from the metric box in the top right hand side of the control panel.

In the result box at the bottom of the page, NFD and MBD are presented as an actual number. NFL is presented in millimeters and tortuosity as a tortuosity coefficient. The results can be exported to an Excel sheet.

When you open the Excel page, the results will be presented in number per millimeter squared for NFD and MBD millimeters per millimeter squared for NFL and tortuosity coefficient for NFT. To make these measurements start by marking each main nerve, then mark each main nerve branch. Finally, trace each nerve in the frame.

A digital pen may be used for better accuracy. Once master, this technique can be done in a minute if it's performed properly while attempting this procedure. It's important to follow the instruction detailed.

After watching this video, we hope that you have a good understanding of how to work and capture images from the movement layer of the corneal with corneal confocal microscopy, and quantify and analyze the images from this layer in patients with peripheral neuropathy, in particular diabetic neuropathy. And don't forget that working with CCM requires expertise and it has been advised that professionals train in eye examination should undertake the procedure.