The protocol allows rapid isolated function of small subpopulations of cells within pancreatic islets of Langerhans. The advantages of this technique include the real-time nature and high statistical power, and hence high reproducibility, of the acquired data. To immobilize the islets for imaging, assemble an imaging chamber for an inverted microscope and place a glass cover slip inside the chamber.
Make sure that the glass chamber interface is watertight and confirm that the cover slip is within the reach of the microscope objective. Next, cut 20 by 20 millimeter squares of fine and coarse mesh and use 45 to 50 micrometer pieces of thick sticky tape to create two spacer walls on a piece of fine mesh. Immerse the coarse mesh and a weight in a 35 millimeter Petri dish of imaging solution, and place the fine mesh under a dissecting microscope.
Turn the fine mesh with the spacer walls upside down and the spacers facing upwards. And use a p20 pipette to transfer several islets between the two spacers. Using watchmakers'forceps, place the mesh upside down inside the imaging chamber of the inverted microscope so that the spacers face downward to sit directly on the chamber cover slip, trapping the islets between the spacers and the mesh in the middle of the cover slip.
Take care that the mesh is well hydrated without containing excessive volumes of solution which would provoke the lateral motion of the sample. Then place the coarse mesh and the weight on top of the fine mesh in the chamber, and add imaging solution to the chamber. Once the islets have been immobilized, select the imaging mode and objective on the inverted microscope and place the chamber with the islets on the temperature-controlled stage of the microscope.
After setting the perfusion, position the inflow lower than the outflow within the chamber and set the outflow flux to be greater than the inflow flux. Ensure that the outflow has minimal contact with the solution so that it removes the solution in multiple sequential small droplets, avoiding long intervals of continuous solution removal. Next, initiate the perfusion with imaging solution containing three millimolar glucose, and select the light path and filters for imaging green fluorophores.
Then run live imaging to set up the imaging parameters and adjust the view to capture the islets of interest. To optimize the signal-to-noise ratio of the image, adjust the excitation light intensity, the exposure time, and the binning, ensuring that the settings allow a distinct visualization of each cell within the islets at the minimal possible light intensity and exposure. Then image the islets at 0.1 to five hertz, checking the quality of the acquired data as it is captured.
When the alpha cell activity is detectable, use an online chart of the signal dynamics within the acquisition software as possible and add adrenaline or glutamate into the bath solution for two to five minutes. A rapid jump in calcium followed by a slowdown or cancellation of the alpha cell oscillations will be observed. Next, add ghrelin, which has been recently reported to activate delta cells selectively.
A rapid reversible increase in calcium will be observed in a small subpopulation of islet cells. Then add 10 millimolar glucose. A coordinated oscillatory response in the beta cell subpopulation will be observed.
Note also the responses of cells that were previously activated by adrenaline or glutamate and ghrelin. After saving the images, import the data into an appropriate data analysis software program and normalize the raw fluorescence intensity data to the initial value of fluorescence. If the cell to cell variability fluorescence intensity data set is still substantial, define a control region for the range of time during which the control solution was applied.
If the control region has a clear non-oscillatory signal, assume that the fluorescence intensity returned to baseline after each application of the control solution. To correct the time lapse data for each cell, split the data into segments separated by the points at which the control solution was added and apply linear correction to each segment. If the control range has clear oscillations or additional factors are present, use a spike detection algorithm.
To measure the frequency of calcium spikes and the response to the addition of agonists and antagonists, split the recording into equal time intervals, count the spikes within the interval, and normalize to the interval duration to compute the time course of the partial frequency. Alternatively, set the threshold and compute the plateau fraction for each of the intervals. The fraction indicates the percentage of time within the interval that the cell spent in the excited state.
Or compute the partial area under the curve for each of the intervals. Unless the lipid composition of the membrane has been affected, islets load fairly well with tropical dyes. The human adenovirus type 5 vector also successfully targets islet cells.
Calcium spiking in alpha cells can be readily detected at low glucose levels, and there is a high cell-by-cell correlation between the activity at low glucose and the response to adrenaline and glutamate. Ghrelin activates some adrenaline responsive cells at low glucose but has no effect on calcium dynamics in most of the cells that are activated by low glucose. When analyzed in terms of partial frequency, adrenaline or ghrelin-stimulated cells display a substantial increase under the all-or-nothing conditions, although the overall changes between basal spiking and the adrenaline effect are subtle.
In contrast, the partial area under the curve is sensitive to the changes introduced by adrenaline in all of the cells even when the basal activity is high. Make sure all the meshes are in contact with imaging solution and take care to position the tissue reasonably densely to avoid air bubbles. The method can be expanded to include the artificial intelligence for differentiating between the cell types.
The pharmacological markers can be replaced by the pattern recognition technology, thereby reducing the recording time.
