Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

The overall goal of this local field fluorescence microscopy imaging technique is to use molecular probes to measure intracellular calcium levels, or membrane potentials from heart regions difficult to access at the whole heart level. This method can help us to answer key cardiovascular questions related to cardiac arrest and ischemia. The main advantage of this technique is that cardiac properties get measured from cellular events in the intact organ where the cells continue to be electrically, mechanically, and metabolically coupled.

First, prepare the horizontal Langendorff apparatus. Load freshly prepared Tyrode solution into the 60 milliliter syringes, and add all the associated tubing of the setup. Once filled, eliminate all of the air bubbles.

Then, equilibrate the loaded solution with pure oxygen gas delivered from a submerged plastic airstone. Next, place a non-absorbable surgical suture around a needle. This will serve as a cannula to retroperfuse the heart when attached to the apparatus.

Next, collect the heart after injecting heparin and euthanizing the animal. First, clean the chest with ethanol. The demonstration is with a mouse, but the same procedure works for parakeet heart harvesting.

Next, using dissection scissors, make an incision in the lower abdomen and then cut up the sides toward the neck. Then, carefully cut open the diaphragm, avoiding the heart. Now, pull back the cut tissue and pin it down.

Next, remove the lungs and surrounding tissues to expose the heart. Once exposed, use tweezers to scoop out the heart without squeezing it. Detach it from the animal by cutting the aorta, and leave as much aorta attached to the heart as possible.

Then, load the heart into a small weigh boat with one milliliter of Tyrode solution. Under a dissecting microscope, quickly remove the blood and the fatty tissue surrounding the heart using tweezers and scissors. Using a non-absorbable surgical suture and a needle, attach the aorta to the horizontal connection of the Langendorff apparatus.

Secure the knot using two fine tweezers. Then, begin the retroperfusion by opening the valve to release the Tyrode solution. Clean the surrounding tissue and suction away debris to maintain the horizontal chamber clean while the heart stabilizes for 10 minutes.

Make the dye fresh. For Rhod-2 AM, add 20 microliters of 20%Pluronic and DMSO to the manufacturer's 50 microgram package of dye. Mix by pipetting, avoiding bubbles.

Then, transfer the liquified dye into a clear glass vial, and add one milliliter of Tyrode solution. Thoroughly dissolve the dye in this mixture using 15 to 20 minutes of sonication. Next, perfuse the prepared dye using a peristaltic pump at room temperature.

First, load the dye in the dye chamber, and then, clamp all the tubing lines connected to the manifold, and keep the clamps above the manifold to prevent backflow. Next, switch on the pump and immediately close the three way valve below the 60 milliliter syringe. Position the small tube attached to the suction end of the pump next to the heart to recirculate the dye after it has been perfused.

Take a small gauge needle and pinch it through the apex to relieve pressure. Then, let the heart perfuse with dye for 30 minutes. During this time, it's very important to evaluate the level of the dye in order for bubbles to not get into the tissue, which would cause ischemia and tissue damage.

After perfusing the dye, retroperfuse the heart with Tyrode solution. Remove the clamp above the manifold. Open the valve, and then allow the heart to stabilize for 10 minutes.

During the retroperfusion, fill the horizontal chamber with Tyrode solution and turn on the Peltier unit to bring the bath temperature to 37 degrees Celsius. To prepare the fiber optic light for the experiment, first place the fiber optic inside a two milliliter pipette. And then attach the pipette to a micromanipulator.

Use the micromanipulator to slightly press the fiber optic against the surface of the lateral ventricle. Next, program a wave generator to provide a square pulse with a width of one millisecond. And set a heart stimulator to be externally synchronized and connected to the wave generator.

At each of the two stimulator outputs, attach a wire with an acupuncture needle soldered at the end. Attach two acupuncture needles to the heart's apex, about three millimeters apart. Only once the needles are placed, start the stimulator, thus avoiding accidental shocks.

Now, to record the epicardial signal, adjust the acquisition frequency in the software to 10 kilohertz. To collect the endocardial signal, follow the same procedure, but use a 23 gauge needle to poke a hole in the lateral ventricle near the septum. And then, using a micromanipulator, position the second fiber optic into the endocardium.

To access transmural function, the morphology of action potentials recorded from the endocardium and the epicardium were compared. Fluorescence recordings were made after treating the heart with di-8-ANEPPS using the CLFFM technique. The action potential was slower in the endocardium, particularly in phase one.

The duration of the action potential was described as the time it takes for the action potential to repolarize to a certain degree. When the durations were normalized and shifted to overlap at the zero phase, they could be compared. There was indeed a significant difference during phase one.

Next, Rhod-2 AM was used to monitor intracellular calcium. In order to compare regional differences in the intracellular calcium, the kinetics of the calcium transience were assessed. By comparison, the time to peak of the transience was about two milliseconds longer in the endocardium.

Overall, the calcium transience from the endocardium had significantly slower kinetics than the epicardium. Once mastered this technique can be used to record intracellular calcium transience and action potentials in regions difficult to access in the heart. The Langendorff portion of the setup makes it very practical to perfuse different chemicals into the heart, and assess the effects on the cardiac properties.