The overall goal of this protocol is to demonstrate the use of second harmonic generation microscopy to visualize and measure the collagen cross-linking effect in scleral tissue as induced by sub-tenon's injection of formaldehyde releasing agent, and as confirmed with differential scanning calorimetry. This method can help answer key questions in the field of therapeutic tissue crosslinking by providing a non-invasive method to evaluate the induced crosslinking effects. The main advantage of this technique is that second harmonic generated signals are highly specific to collage fibrils, and as such, no special tissue preparation, such as antibody staining, is required.
We first had that idea for this method when we read about the procedure being applied to therapeutic corneal tissue crosslinking. Visual demonstration of this method is critical as successful second harmonic generation microscopy requires careful placement of the tissue and setting correct imaging conditions to prevent tissue damage and artifacts in signal measurement. Begin the treatment with sub-tenon's injection in the rabbit's eyeballs as described in the text protocol.
Show here is a schematic representation of the sub-tenon's injection site. Select an optimally sized speculum according to the size of the eye. After three and a half hours of incubation, retract the eyelids using the eyelid speculum to optimize access to the globe.
Separate the conjunctiva surrounding the limbus and cut the extra ocular muscles at their sites of scleral insertion. Cut the upper part of the sclera using scissors or a blade to create a one by one centimeter area with the site of injection located centrally. Scrape off the remaining retinal and choroid layers and wash twice with fresh PBS, each time leaving the pieces in solution for approximately 10 seconds.
Place the tissue in one milliliter tubes filled with PBS solution for transportation to the imaging facility. To maximize the signal and resolution when performing SHG microscopy, use an objective lens optimized to transmit infrared light, and with a high numerical aperture. Adjust the correction collar of the lens to match the depth of the sample.
In this case, 0.17 millimeters. Mount the 25 times objective lens on the microscope and add a generous amount of water based immersion gel to cover the lens surface. It is important to perform the tissue imaging within 30 minutes form mounting the sample on the microscope in order to maintain the tissue hydrated and prevent drying of the immersion gel between the lens objective and the slide, as it would result in changes in the SHG signal.
Assemble the cell chamber by placing a 25 millimeter round cover slip on the bottom part of the chamber. Place the piece of sclera tissue on the cover slip and add 50 to 100 microliters of buffer. Cover the tissue with a second round cover slip and screw on the top part of the chamber without pressing or flattening the tissue.
Then, mount the cell chamber with the tissue sample on the microscope stage. Position the stage and adjust the height of the objective such that the lower surface of the sample is in focus as determined by bright field inspection through the eye piece. Turn off all lights except the computer monitor, and block as much light from the monitor as much as possible with aluminum foil sheets draped on the microscope stage.
In the software, switch off the Eye Port mode. In the TI Pad panel, check that the lens definition is correct. In the A1 Compact Graphical User Interface panel, choose the infrared laser for imaging, select the Non Descanned Detectors, and choose the DAPI channel that is equipped with a 400 to 450 nanometer band pass filter.
In the A1 Multi Photon Graphical User Interface panel, set the wavelength of the infrared laser to 860 nanometers and open the shutter. Wait until emission wavelength is changed as indicated by the steady green light. To set the laser scanning conditions in the A1 Compact Graphical User Interface panel, select Galvano scanner, unit directional scanning, a frame size of 1024 by 1024 pixels, pixel dwell time of 6.2 microseconds, and line averaging of 2X.
Then, turn on live imagining with the find mode, and set the imagine conditions in the AI Compact Graphical User Interface panel by adjusting the laser power and detector gain. Set laser power between 50 milliwatts and 150 milliwatts, that is, between two percent and six percent for a laser at 860 nanometers. Above this range tissue damage can occur.
Open the Lookup table panel which displays a histogram of pixel intensity values in the current image, and further adjust the gain of the detector in the HV labeled bar to maximize the range of pixel values while avoiding saturation. Use the sample with the highest SHG signal to find optimal laser and gain settings. Maintain identical settings for all samples to be compared in one experiment.
This practice ensures that no sample in the experiment will saturate the detector. To scan the tissue are in preview mode, open the XYZ overview tool. Set the imaging to lower resolution to speed up the acquisition of images in this mode.
In the XYZ overview panel, right click on the current position and select scan five by five, three by three, or single view capture. After scanning is finished, move to a new location by double-clicking on an area outside the current previewed area. Turn on the Live Scan mode, then bring the tissue into focus as different regions of the tissue can have slightly different positions in the axial direction.
Capture a new five by five, three by three, or single fields of view. Repeat this process until a large area of the tissue has been previewed. In the XYZ overview tool, double-click a position where the collagen fibers are seen in the entire field of view in order to move the stage to that particular location.
Set imaging to high resolution, 1024 by 1024 pixels and 2X line averaging. Turn on Live Find mode, adjust the position of the objective such that the bottom plain is in focus, and in the TI Pad, use the Z Drive to move the optical plane 10 micrometers above this bottom layer. Acquire an image using the capture button, save the location with the plus button in the XYZ review tool.
Double-click a new location and repeat the depth adjustment and image capture process. For each piece of tissue, capture 10 images. The fields of view should not overlap as shown by the saved locations in the XYZ overview tool.
Finally, place the scleral pieces in a one milliliter tube with PBS immediately after microscopy is completed in preparation for subsequent differential scanning calorimetry analysis. To begin, cut out the central part of the control and treated sclera with scissors. Dry each scleral square with an absorbent tissue and lay it flat on the bottom of a DSC pan using toothed forceps.
With the tissue inside and the lip crimped and covered, weigh the pan in order to obtain the tissue wet weight. Then, place the weighed sample in its designated location on the DSC autosampler tray. Create a method using instrument managing software, specifying the weight of the tissue and run the thermal analysis using the perimeters listed in the text protocol.
Once completed, analyze the data for each sample by extracting the transition temperature peak at which thermal denaturation occurs using the instrument imaging software. SHG images of sclera were analyzed in Fiji for mean pixel brightness. When an image is opened and the analyze menu is selected followed by measure, a table with results opens containing the mean brightness of the signal.
Alternatively, in the Analyze menu, Histogram is selected to create a histogram of pixel intensity values that contains information about the mean brightness and variation of the signal in the image. This measurement was repeated for all the images from the control and cross-linked samples. Representative SHG images of sclera with accompanying histogram analysis, include mean brightness, are demonstrated here.
The control sample is characterized by a wavy arrangement of fibrils. Shown here is a sample cross-linked with 400 millimolar SMG with increased pixel intensity revealing collagen fibers that appear straighter. Results of the thermal analysis show the higher peak of denaturation temperature and confirm the cross-linking effect.
The control sample, and a 400 millimolar cross-linked sample denaturation curve are shown. After watching this video you should have a good understanding of how to prepare a cross-linked sample, and how to perform SHG microscopy on scleral samples. Generally, individuals new to this method with struggle with precise injection in the sub-tenon space, surgically extracting the square around the injection site and setting the images conditions to scan the tissue in the preview mode.
Following this procedure, other methods, like autoluorescence can be performed in order to answer additional questions, such as elastin analysis including quantitation and morphology. The implications of this cross-linking agent delivery technique extend toward therapy of high myopia and posterior staphyloma, because it provides easier access to the posterior pole for the purpose of scleral stabilization. Implications of this microscopy technique extend toward diagnoses of high myopia and complications caused by collagen weakening by measuring state of collagen cross-linking in the diseased sclera, or by measuring the outcome after the desired cross-linking procedure is completed.
So, this cross-linking method and second harmonic generation microscopy can provide insight into scleral cross-linking, it can also be applied to other tissues such as cornea, skin, cartilage, tendon, heart valve and blood vessels.
