Imaging Calcium Dynamics in Pancreatic Cells: A Technique to Study Real-time Changes in Cytosolic Calcium Concentration in Pancreatic Islet
Transcrição
– Pancreatic islets are a cluster of hormone-producing pancreatic cells that play a crucial role in maintaining glucose homeostasis. Elevated glucose concentration triggers the closure of potassium ion channels, preventing potassium ion efflux, leading to membrane depolarization. In consequence, voltage-dependent calcium ion channels open, allowing the influx of calcium ions. This increased cytosolic calcium concentration stimulates the release of hormones like insulin. In real-time, to image cytosolic calcium dynamics, transfer a few islets loaded with suitable calcium indicator dye between the spacer walls of a preassembled fine mesh.
Position the mesh upside down on a coverslip set inside the imaging chamber of an inverted microscope. Place a coarse mesh and weight to immobilize the cells. Add imaging solution into the chamber. Next, set up a perfusion system to introduce glucose or a test hormone of interest. Within the cells, preloaded fluorescent calcium indicators report any increase in cytosolic calcium concentrations. Upon binding to calcium ions, the indicator molecules exhibit a substantial increase in fluorescence emission. In the following protocol, we will show the real-time imaging of calcium dynamics in murine pancreatic islets in response to glucose perfusion.
– To immobilize the islets for imaging, assemble an imaging chamber for an inverted microscope and place a glass coverslip inside the chamber. Make sure that the glass chamber interface is watertight and confirm that the coverslip is within the reach of the microscope objective. Next, cut 20 X 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 watchmaker's 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 coverslip, trapping the islets between the spacers and the mesh in the middle of the coverslip.
– 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 perifusion, 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 of waiting long intervals of continuous solution removal. Next, initiate the perifusion with imaging solution containing 3 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 5 Hertz, checking the quality of the acquired data as it is captured.
