The ACE is an optimal imaging sights. It supports to repeat it in long-term in vivo imaging of islet cells in order to study their function and viability in a grafting situation. It can also be extended to study other aspects under physiological and pathological conditions in real time.
The advantage of the ACE technique lies in the fact that the same individual human pancreatic islets can be followed noninvasively with sub-cellular resolution for an extended period of time. Success rates of beta cell replacement therapy continue to improve. However, evaluating the efficiency of islet grafting and survival in vivo, remains still challenging.
This method could help assess and optimize the outcomes of islet transplantations. This approach is not restricted to pancreatic islets only. It is also well-suited to study tissues affected.
For example, in diabetic complications, like kidney glomeruli or liver pieces, just to name a few. To begin place the mouse on a warm heating pad position it in the head holder and put on the nose mask. Use the thumb and index finger to lift the head slightly.
Fasten it with the metallic pieces on the sides. Making sure that the ear pieces fix the head directly below the ears. Subcutaneously inject buprenorphine solution into the back of the mouse.
Gently retract the eyelids of the eye to be transplanted using blunt forceps. Pop the eye out and loosely fix it with a pair of tweezers covered with a polythene tube. After transferring the eyelids to a Petri dish with PBS, pick up approximately 20 to 30 islets in the eye cannular.
Using a 25 gauge needle level upwards, carefully insert the tip into the cornea and make a single lateral incision. Carefully lift the cornea with the cannula preloaded with islets, and slowly release the islets into the eye. Slowly retract the cannula and apply eye gel to the eye.
After 10 minutes, remove the forceps, holding the eyelid and put the eye back to its normal position. For imaging of implanted human islets with two photon microscopy, place the head holder platform under the microscope and administer eye gel onto the eye as an immersion liquid between the cornea and the lens. Position the eye under the objective and image the implanted human islets as described in the text manuscript.
To remove the autofluorescence, go to the image processing tab, choose channel arithmetics and type channel one minus channel two. This creates a new channel four. Rename it vasculature.
Repeat this process and type channel three minus channel two to create a new channel five and rename it tomato all. To define the islet manually create a new surface and choose edit manually in the wizard. Keep the pointer in select mode and in 3D view.
And click volume to visualize sections. In the drawing tab choose contour and click draw to start drawing contours around the islet border, starting in slice position one. Then move to a new slice position and repeat drawing contours.
Finish with the last slice on the top of the islet and end by clicking the create surface tab. Next segment, islet vasculature and islet tomato fluorescence using islet mask. Choose the previously defined islet mask object, go to the editing tab and click the mask all tab, which opens a new window.
Choose the vasculature channel in the channel selection dropdown menu and activate options duplicate channel before applying mask. Constant inside, outside and set voxels outside surface to 0.000 which creates a new channel six. Rename this channel islet vasculature.
Repeat the previous steps and choose the tomato all in the channel selection dropdown menu, to create the new channel, rename it, islet tomato. Then create a new surface in the scene menu and choose automatic creation in the wizard. Set the source channel to the previously created islet vasculature and choose background subtraction.
Optionally use filters. For example choose volume and adjust the filter in the window, which can remove selected service objects. Finish the wizard and name the new surface object, islet vasculature.
In the islet vasculature service object go to the editing tab and click the mask all tab, which opens a new window. Choose the islet tomato channel in the channel selection dropdown menu and set voxels outside surface to 10.000 which creates a new channel. Rename this channel islet tomato vasculature.
To segment tomato capsule fluorescent signal choose channel arithmetics in the image processing tab and type channel seven minus channel eight creating the new channel. Rename it tomato capsule. Create a new surface as previously described and choose source channels, islet tomato vasculature or tomato capsule in the wizard.
Then perform background subtraction. Open the islet backscatter file and create a new surface. In the wizard, choose automatic creation and define region of interest.
If needed, adjust the threshold in absolute intensity. The surface object can be clicked on or off, to crosscheck with corresponding channel intensity. When finished close the wizard.
Finally, perform quantification, select a created surface object in the scene menu, and go to the statistics tab. To retrieve detailed volume data, choose the detailed tab and select specific values and volume from the dropdown menu. To retrieve total volume value go to the detailed tab and choose average values.
This protocol was used to transplant non labeled human islets into the interior chamber of the eye of eight-week old female red fluorescent recipient mice. Interactive imaging software was then used to extract quantitative data from the images. In vivo fluorescens imaging, the human islet grafts was compromised by significant interference from tissue autofluorescence which was not observed in mice islet grafts.
To improve the signal to noise ratio, the autofluorescence channel was subtracted. Then the islet boundary called islet mask and the corresponding channels were defined manually. Finally, the segmentation of the tomato signal into the eyelid vasculature and eyelid capsule and final surface rendering was used to extract quantitative data.
Here as a representative longitudinal imaging session of the same human islet graft at two weeks, two months, five months and eight months post transplantation. MIP images of originally recorded raw data, processed images after islet autofluorescence removal and a segmented islet objects are shown. When performing the eyelid injection, make sure to preload the islets in a small volume and use flexible twin light arms on your stereo microscope, to highlight the eye chamber space in order to successfully inject the islets into the ACE.
Following the in vivo imaging the eyes can be fixed and further investigated for antibody staining and conventional histology. This technique is very useful for functional studies of individual human islets in real time and under physiological conditions, especially in the context of vascularization, viability and metabolic studies.
