This method can help understand important questions in the beta-cell field. Such as the function heterogen IT among individual beta-cells within an islet. The main advantage of this technique is the spatial and temporal resolution provided by live imaging of the beta-cells.
We can capture the calcium oscillations within individual beta-cells. After euthanizing a fish according to the text protocol, transfer it to a Petrie dish containing HBSS solution with calcium and magnesium. Then under a stereo microscope equipped with a fluorescence lamp and a red filter cube, use sharp forceps to cut the skin from the mouth to the anal fin.
Peel away the cut skin on the right side to expose the abdomen, which will expose the internal organs. Then using the red fluorescence to identify mKO2 expression in beta-cells, locate the islets. Clean the primary islet by carefully removing the surrounding tissue, such as liver and adipocytes.
Take precautions not to injure or poke the islet. Next pipette a 30 microliter drop of HBSS onto the center of a glass bottomed dish. Then transfer the dissected islet into the drop.
With HBSS, carefully wash the islet once then use 30 microliters of fibrinogen working solution to wash it once. Avoid drying the islet during the washing steps which could result in cell death. Slowly and gently add 10 microliters of 10 units per milliliter of thrombin solution to the islet.
Leave the islet and the dish untouched for 15 to 20 minutes. Observe that the fibrinogen thrombin drop will become viscous and stable. Sufficient time needs to be given for the thrombin to polymerize in the fibrinogen solution.
Otherwise the mold will not provide stability during the imaging session. To carry out ex vivo live imaging of GCaMP fluorescence, add 200 microliters of HBSS on top of the mold and carefully place the dish on the plate holder of the confocal microscope. Then with a 20X 0.8 NA error objective and the bright field option, locate the islet.
Using the filter for red fluorescence to view the nuclear mKO2 fluorescence in beta-cells focus on the islet. Individual nuclei should be clearly visible. Locate a clear imaging plane by manually moving through the thickness of the islet along its Z axis.
Ensure that the imaging plane contains 50 to 100 beta-cells for imaging and the brightness of the nuclear mKO2 fluorescence is uniform, especially in the center of the islet. Next in the smart setup menu, setup a sequential acquisition for GCaMP6 and mKO2 fluorescence using the following settings. For GCaMP6 choose an excitation of 488 nanometers and an approximate emission of 500 to 555 nanometers.
Under false color select GFP. For mKO2 choose an excitation of 561 nanometers and an emission of 570 to 630 nanometers. For the false color select mCherry.
In acquisition mode, set the image resolution to 1, 024 by 1, 024 pixels, the speed to 10, and averaging to one. Initiate a continuous recording by selecting the option for Time Series and setting the duration to 500 cycles with approximately two seconds acquisition time per frame. Keep an eye on the imaging cycle.
After the first 50 frames without perturbing the image acquisition, gently pipette five microliters of 200 millimolar D-glucose solution on top of the gel holding the islet. Then acquire 150 frames at 10 millimolar glucose. The first 50 frames of the Time Series corresponds to beta-cell activity at five millimolar glucose.
This is the basal activity. A responding beta-cell will show waxing and waning of green fluorescence with time. At 200 frames, increase the glucose concentration to 20 millimolar by gently adding 10 microliters of 200 millimolar D-glucose.
Then acquire 150 frames at the 20 millimolar concentration. To open the image file in FIJI, select Plugin, LSM Toolbox, show LSM Toolbox. In the LSM Toolbox click Open LSM and select the image file.
Under Tools in the Analyze menu open the ROI Manager. With the polygon selection tool located in the toolbar, manually draw the ROI. Draw the ROI in the red channel so that it covers an area larger than a nucleus to include some of the cell's cytoplasm.
Ensure that the ROI position is consistent between frames and adjust the position if necessary. Add the selected ROIs to the ROI Manager by clicking on the Add Button. Select and add multiple ROIs to obtain data on multiple cells.
Next from the Analyze menu, select Set Measurements. Then select Integrated density for specifying extraction of total fluorescence intensity within the area. Shift to the green channel containing the GCaMP fluorescence and in the ROI Manager select Multi Measure.
This will provide the intensity measurements for the cells throughout the Time Series. Save the FIJI output as a comma separated text file. From the LSM Toolbox, obtain the time stamps of the image frames.
Use Apply Stamps, Apply t-stamps, File Name, Dump to textfile, to obtain the time stamps. Save the time stamps using the Save as option or copy them into the spreadsheet. Upon compiling the intensity values for all the cells, perform the analysis one cell at a time or automatically.
Refer to the text protocol for additional details. In this experiment, individual primary islet beta-cells from 45 DPF transgenic line expressing nuclear mKO2 and GCaMP6 specifically in beta-cells, were stimulated with a glucose ramp and then depolarized with potassium chloride. The activity of the cells was analyzed.
Using FIJI and data analysis software, GCaMP6 fluorescence intensity of individual beta-cells was extracted and normalized. As seen from this trace of fluorescence intensity, individual beta-cells display oscillations in GCaMP6 fluorescence when stimulated with glucose, and the addition of potassium chloride stabilizes the fluorescence. The technique provides a cellular resolution of the beta-cell's glucose responsiveness and a window into their functionality.
Once mastered, this technique can be performed in 45 minutes if done correctly. While performing this procedure it is important to verify the viability of the beta-cells. Following this procedure other methods, like genetic manipulations, can be performed to understand the role of specific genes on beta-cell function.
After watching this video you should have a good understanding of how to measure glucose response within individual beta-cells.
