Imaging Calcium Dynamics in Pancreatic Cells: A Technique to Study Real-time Changes in Cytosolic Calcium Concentration in Pancreatic Islet

1. Load the dye or express the sensor

1. Prepare the trappable dye.
   1. Dissolve the aliquot (normally, 50 μg) of the trappable dye (e.g., Fluo-4) in DMSO to a stock concentration of 2 mM. Add pluronic acid to a final concentration of 1% (using a 20% stock in DMSO), to improve the solubilization of the dye.
   2. Aliquot the dye in small PCR tubes (2 μL into each). The dye can be stored frozen (-20 °C) for several weeks.
2. Alternatively, prepare the recombinant sensor.
   1. Distribute the sensor (e.g. adenoviral vector encoding GCaMP6f) into 10 μL aliquots and store at -80 °C.
   2. (Optionally) pre-test the titer of the viral stock by infecting islets (as below) using several serial dilutions, to reveal the optimal concentration for infection.
      NOTE: Recombinant sensors encoded by different vectors (lentivirus, BacMam, AAV) require a different infection protocol. Make sure to check this with the vector provider and optimize the working ratio for your needs. The "working" stock of the adenovirus can be stored at -20 °C and frozen/thawed several times. Excessive freeze-thaw cycling reduces the effective titer of the virus.
3. Prepare the imaging solution.
   1. Make up the imaging solution, mM: 140 NaCl, 4.6 KCl, 2.6 CaCl₂, 1.2 MgCl₂, 1 NaH₂PO₄, 5 NaHCO₃, 10 HEPES (pH 7.4, with NaOH).
   2. Make up a stock of glucose (0.5 M) and mannitol (0.5 M) in the imaging solution. The stock can be stored in the fridge (4 °C) for several weeks.
4. Load the trappable dye.
   1. Make up the dye working solution by dissolving 2 μL of the dye in 600 μL of the imaging solution containing 6 mM glucose. The solution can be heated up or vortexed to improve the solubilization.
   2. Load the islets isolated into the working solution of the dye. The loading can be done using a multiwell plate or a Petri dish. In the latter case, place a 100 μL droplet of the working solution on the bottom of a non-adhesive Petri dish (35- or 60-mm) and pipette 10-30 islets into the droplet.
   3. In case of different groups of islets (e.g. wild-type/knock-out), arrange multiple wells and multiple droplets so the loading can be done simultaneously.
   4. Incubate the islets in the dye working solution at room temperature in the darkness for 70-90 minutes. Do not over-incubate.
   5. Check the loading under the fluorescence microscope; the islets should gain mild fluorescence, with some cells being brighter than the rest. Rounding up of cells and nuclear localization of the dye are signs of overloading.
   6. Transfer the islets into dye-free imaging solution containing 6 mM glucose. The islets can be used for imaging immediately, but optionally the dye can be left to de-esterify for another 10-15 minutes. Islets will retain the dye for several hours and therefore can be used for imaging in several shifts.
5. Alternatively, infect the islets with the recombinant vector.
   1. Plate the islets in droplets in RPMI culturing medium (RMPI1640 (see Table of Materials), supplemented with 10% of fetal calf serum, 100 units/mL penicillin, 100 μg/mL streptomycin. Dispense the medium into two 60 mm plastic Petri dishes (not treated with any adhesive), and keep the dishes in the incubator (37 °C, 5% CO₂, absolute humidity)). (e.g., 30 μL), to minimize the volume of vector needed.
   2. Add the vector at a ratio of approximately 10⁵ infectious units/islet that should ideally result in multiplicity of infection >2. Ideally the ratio should be optimized to the minimal ratio that would provide expression in the peripheral layer. Pre-titration (step 1.2.2) may help.
   3. Introduce 20-50 islets into the droplet and culture for 8-48 hours. (Ideally, overnight). Islets should develop a faint green fluorescence in most of the cells, without changes in cell morphology.
      NOTE: The success of the infection and expression depends on the time of exposure to the virus solution. Ideally, the virus should stay in the solution overnight but can be optionally removed after as little as 15 minutes. However, infectivity, and therefore expression, is likely to be dramatically lower.

2. Imaging Ca²⁺ dynamics

1. Immobilize the islets under the (inverted) microscope.
   1. Assemble the imaging chamber for inverted microscopy. Position the glass coverslip (thickness 1 or 1.5) inside the chamber and make sure that the glass-chamber interface is water-tight. Check that the coverslip is within the reach of the microscope objective (critical for the case of a bulky high-NA objective).
   2. Prepare the immobilization accessories. Cut small rectangles (20 mm x 20 mm) from the fine mesh and the coarse mesh. Introduce two spacer "walls" on the fine mesh using a 45-50 μm thick sticky tape. Use double layers of the spacer if the size of the islets to-be-imaged substantially exceeds the conventional 100 μm.
   3. Immerse the meshes and the weight into the imaging solution using a 35 mm Petri dish. Make sure that the plastic and the metal are wet.
   4. Under a dissection microscope, turn the fine mesh with the spacer "walls" upside down, the spacers facing upwards. Pick several islets loaded with the trappable dye or expressing the recombinant sensor with a P20 pipette and gently position them on top of the fine mesh, between the two spacers. Ensure that the mesh and the washers do not contain excessive amounts of the imaging solution on them.
   5. Pick up the mesh with the islets, using watchmaker's forceps, and position it upside down inside the imaging chamber, so that the spacers face downwards and sit directly on the chamber coverslip. Ensure that the islets are trapped between the spacers and the mesh, in the middle of the coverslip.
   6. Position the coarse mesh and the weight on top of the fine mesh within the chamber. Introduce the imaging solution into the chamber. Ensure that the islets are immobilized and ready to be imaged. Avoid excessive shaking of the chamber (small perturbations like carrying the chamber to the microscope and inserting into a heated stage are acceptable).
      NOTE: A similar immobilization arrangement can be applied for an upright system.
2. Set up the microscope.
   1. Choose the imaging mode and the objective, position the chamber with the islets from step 2.1 on the temperature-controlled stage of the microscope.
      1. Set the temperature control (ideally, between 30 °C and 36 °C) and the perfusion. For an inverted system, position the inflow lower than the outflow within the chamber, and set the outflow flux to be greater than that of the inflow (which is typically achieved by using a tubing of a wider inner diameter on a peristaltic pump).
      2. Ensure that the outflow has a minimal contact surface with the solution, so that it removes the solution in multiple sequential small droplets, avoiding long intervals of continuous solution removal. The latter is the major source of artefact in time-lapse imaging of the periodical signals as they appear as regular periodic intensity oscillations of every imaged pixel and are frequently interpreted as "slow waves".
      3. Initiate the perfusion with the imaging solution containing 3 mM glucose.
   2. Choose the light path and filters for imaging of the green fluorophores; excitation between 470 and 500 and emission between 505 and 550 would work for each of them.
   3. Run live imaging to set up the imaging parameters. Adjust the view to capture the islets of interest.
   4. Optimize the signal-to-noise ratio of the image. To that end, adjust the excitation light intensity, the exposure time and the binning. Ensure that the settings allow a distinct visualization of each cell within the islet at the minimal possible light intensity and exposure.
   5. Perform image acquisition. Depending on the task, images can be taken at 0.1 to 5 Hz. This is well below the Nyquist criteria for the fast Na⁺-driven oscillations in α- and δ-cells (>300 Hz), which means that the data is undersampled by default. However, increasing the acquisition frequency to match this demand is not feasible in multicellular/multi-islet imaging with a large field of view. GCaMP can be imaged faster, whereas Fluo4 will inevitable bleach under fast acquisition conditions.
      NOTE: Given that [Ca²⁺]_cyt oscillations in the islet cells are driven by electrical activity, using low acquisition rates may sound counterproductive. In reality, however, acquisition rates at around or above 1 Hz may be sufficient for resolving β-cell spiking behavior, whereas the threshold for detection of sodium channel-driven oscillations in α- and δ-cells is well above 300 Hz. Whether the α- or δ-cell [Ca²⁺]_cyt oscillations are acquired at 1 Hz or 0.1 Hz, they will be severely undersampled and reflect Ca²⁺ handling by the cell rather than electrical activity.
      1. Check the quality of the acquired data: at 3 mM glucose, α-cell activity should be clearly visible/detectable. Ensure this is the case and proceed to full-scale time-lapse imaging.