Understanding pancreatic beta cell proliferation and eyelet growth is an important aspect of diabetes research. In this video article, imaging and analysis of a whole mouse pancreas is demonstrated using mice with GFP expressing beta cells, the pancreas is removed from a euthanized mouse and flattened on a glass slide. The pancreatic tissue is cleared by sequential treatment of paraform aldehyde Triton, X sucrose, and glycerol using fluorescent microscopy.
An image or virtual slice of the pancreas is acquired. The image is then analyzed to determine the number, size and distribution of eyelets within the pancreas. Hi, I'm German Kilimnik from Dr.RA's lab in the Department of Medicine at the University of Chicago.
I'm Abraham Kim, also from the HAR Lab. Today we will be showing you a procedure for the insight to imaging and quantification of all the beta cells in a mouse pancreas. We use this procedure in our laboratory to study mouse outlet structure, development and formation.
So let's get started. Begin this procedure by applying 95%ethanol to the mouse's skin to avoid fur contamination. Then lift the mouse's skin on the ventral side and make an incision near the lower abdomen.
Cut along both sides of the body in a U-shape. To expose the organs, place the euthanized mouse with GFP labeled beta cells under a dissection microscope. Using forceps, remove the entire pancreas with the duodenum in the spleen still attached and place the organs on a glass slide next, remove the duodenum by cutting its connective tissue along the side where it's attached to the pancreas.
Cut and remove the spleen and the white colored excess fat from the pancreas. Continue working until the pancreas is completely isolated on the slide. Then weigh the pancreas on the slide.
Next, place a cover slip over the pancreas so that it's entirely covered. Use a weight to flatten the pancreas between the slide and the cover slip. Allow about 10 minutes for the pancreas to flatten.
After 10 minutes has passed, remove the weight, submerge the slide in 4%param, aldehyde or PFA. Then place the slide at four degrees centigrade overnight. The next morning, remove the cover slip and expose the whole pancreas to PFA at four degrees centigrade.
After eight hours, examine the pancreas. If it is not well fixed and appears raw, place it back in the PFA and continue to fix overnight following fixation. Transfer the slide to the container with 1%Triton X 100 in PBS and store it at four degrees centigrade overnight.
The next day, transfer the slide to a container of saturated sucrose. Incubated four degrees centigrade for one to two days. Then transfer the slide to a container of 100%glycerol for optimal imaging resolution.
The slide should remain in glycerol for one to three days until the tissue has cleared and appears transparent. The pancreas shown on the right is not fully cleared. Store slides in a glycerol solution and place them in a dark, cool area to preserve the organ and its fluorescent signals.
Although fluorescent signals may be persistent for weeks and even months, imaging should be performed as soon after tissue clearing is possible. For optimum resolution for imaging, use forceps or gloved hands to take the slide out of the glycerol. Then prepare the slide for imaging by wiping off excess glycerol and cleaning the glass with ethanol.
Open the stereo investigator software for imaging mice pancrea older than three weeks as shown here, select the two x objective lens. Visualize the image and stereo investigator by clicking on the image toolbar at the top of the screen and selecting live image. When the image appears, select an exposure time that clearly shows the GFP tagged beta cells.
Under exposing the image excludes smaller eyelets and beta cells while overexposing the image creates additional false signals. Click anywhere on the screen to establish a reference point and proceed to draw a contour around the entire pancreas. To initiate the virtual slice process, click on image, then acquire virtual slice.
Using the focus knob, determine and select the best focal plane that is the plane with the highest number of visible and focused eyelets and beta cells. Once the plane is chosen, stereo investigator automatically captures the image displayed on the screen. Virtual slice imaging uses an epi fluorescent configuration.
The widefield filters allow the camera to capture all signals present at a certain depth, including those not perfectly in focus. The final virtual slice is an integrated two dimensional image that is not a maximum projection from the three dimensional reconstruction of the whole pancreas. The average virtual slice scanning time is 30 minutes per sample, but it can take up to an hour to scan a large pancreas with the motorized stage.
The virtual slice feature sequentially captures each optical panel as a distinct image within the drawn contour and then combines all of them as one merged image. Save the image. Begin analysis by opening up the image with image J when the image finishes loading and appears on the screen.
Open and run the virtual slice analysis macro, which is provided as a supplementary file. With this video, follow the step-by-step instructions provided by the macro. Eliminate undesired artifacts created by light scattering with the select area tool.
Check and subtract unnecessary background from the image. Using the line tool determine the radius of the largest eyelet. The rolling ball feature then automatically uses the largest radius to remove autofluorescence and scattered light.
Drag the slider in the threshold window to capture the fluorescence signals of the small eyelets and clusters of beta cells. Determine the most appropriate low threshold. Next, drag the slider in the threshold window to capture the fluorescent signals of eyelets.
Select the most appropriate high threshold. Finally, run the quantification of the TU slice by clicking yes when the analyze window appears, which will measure and produce a list of eyelet area perimeter circularity and rez diameter images of individual optical panels of a male mouse pancreas at 10 weeks taken under a two x objective are shown here. The images include the entire distribution of eyelets, including small clusters of beta cells, less than 10 cells.
Here a unified virtual slice with the dorsal pancreas on the right and the ventral pancreas on the left is shown. The scale bar shown is five millimeters. Shown here is a mask of the fluorescent particles in the virtual slice.
Blue indicates regions with low fluorescence intensity, green intermediate intensity, and red high intensity fluorescence. This figure shows quantification of individual eyelets clusters of beta cells. Four parameters were taken for each structure area, perimeter circularity, a degree of roundness where the number 1.0 represents a perfect circle and ferrett's diameter the longest distance within an area.
Note that number 7 0 6 displays the analyses resolution with an area of only a few beta cells. Watershed segmentation detects groups of adjacent eyelets such as the one shown and appropriately distinguishes them as distinct eyelets. Each eyelet, including small clusters of beta cells, is numbered with its information detailed in a corresponding chart.
Here is a three-dimensional plot of the virtual slice analysis with ES of area circularity and res diameter. Each dot represents an eyelet. We've just shown you how to prepare an image.
All GFP labeled beta cells in a mouse pancreas. When doing this procedure, it is important to be mindful of the sensitivity of the mouse pancreas. So that's it.
Thanks for watching and good luck with your experiments.
