Name: local field fluorescence microscopy: imaging cellular signals in intact hearts
Uploaded: 2017-03-08T13:00:00
Description: Watch this Scientific Journal Video about Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts at JoVE.com

We thank Dr. Alicia Mattiazzi for critical discussion of the presented work. This work was supported by a grant from NIH (R01 HL-084487) to ALE.

This protocol and all mice handling was approved by the UC Merced Institutional Animal Care and Use Committee (No. 2008-201). Experiments with parakeets were conducted in 1999 according to general policies for animal use established by the scientific commission of the Venezuelan Institute for Scientific Research (IVIC).
1. Langendorff Set Up Preparation
<ol>
	<li>Prepare Tyrode solution containing the following solute concentrations in mM: 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2<...

<ol>
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This paper is centered in describing local field fluorescence techniques to evaluate the function of cardiac myocytes ex vivo. The study of these cells in a coupled environment is not only more physiological, but is also highly appropriate for assessing organ-level pathologies. The cellular events underlying excitation-contraction coupling (ECC) can be evaluated at the whole organ level with the use of molecular probes that monitor intracellular Ca&#8203;2+ dynamics (Rhod 2 cytosolic Ca2+, ...

The heart is the central organ of the cardiovascular system. The heart&#39;s contraction is initiated by an increase in intracellular [Ca2+]. The relationship between electrical excitability and changes in intracellular Ca2+ release has been historically studied in enzymatically dissociated cells1,2. However, cardiac cells are electrically, metabolically and mechanically coupled3,4. When isolated, the myocytes are not only physically uncoupled, but myocytes from different layers are mixed during the dissociation5. Furthermore, despite the enormous advantages that have emerged from the study of isolated cells under voltage clamp conditions,6,7,8 the intrinsic nature of the heart as an electrical syncytium always poses the question of how functionally different are dissociated cells from those present in the tissue3.
In this manuscript, we describe the advances obtained in the knowledge of cardiac physiology by the use of local field fluorescence microscopy (LFFM) techniques in the intact heart. LFFM uses fluorescent indicators to measure multiple physiological variables such as cytosolic Ca2+, intra sarcoplasmic reticulum (SR) Ca2+ and membrane potential. These measurements can be obtained simultaneously and in conjunction with ventricular pressure9,10, electrocardiograms9, electrical action potentials (APs), ionic current recordings and flash photolysis of caged compounds4,11. In addition, these measurements can be obtained by pacing the intact heart at higher frequencies closer to physiological rates. Although several articles9,11,12,13,14 have been published by our group using LFFM techniques, the presumption of technical complexities associated with this technique has prevented its massive use in studying ex vivo physiological phenomena in the heart and other organs.
The LFFM technique (Figure 1) is based on epifluorescence measurements obtained using a multimode optical fiber in contact with the tissue. Like any contact fluorescence imaging technique, the optical resolution depends on the diameter and the numerical aperture (NA) of the fiber. A higher NA and smaller diameter of the fiber will increase the spatial resolution of the measurements. NAs and fiber diameters can range from 0.22 to 0.66 and from 50 &#181;m to 1 mm, respectively. Increasing the NA will improve the signal to noise ratio (S/N) by accepting photons arriving from a larger solid angle. In order to act as an epifluorescence device, the light beam is focused into the optical fiber with an aspheric lens or an epifluorescence objective where the NA of the lens and the fiber match. This matching maximizes the energy transfer for excitation and for collecting back the photons emitted by the fluorophore.
In order to excite the exogenous fluorescent indicators loaded in the tissue, different light sources and illumination modes can be utilized. Our pioneering studies using the pulsed local field fluorescence microscopy3,12 (PLFFM) employed a low-cost picosecond laser (Figure 1a, PLFFM). This type of light source has the huge advantage of exciting a big fraction of fluorophore molecules under the illumination area without substantially bleaching the dye due to the short pulse durations12. Additionally, the use of ultrashort pulses allowed the assessment of the fluorescence lifetime of the dye12. The fluorescence lifetime is a property that can be used to quantify the fraction of dye molecules bound to Ca2+. Unfortunately, the temporal jittering of the pulses and variations in amplitude from pulse-to-pulse limit the application of this experimental strategy to cases where the change in fluorescence produced by the ligand binding to the dye is large.
Continuous Wave (CW) lasers are usually used as the main illumination source in LFFM (Figure 1b, CLFFM). The laser beam can continuously illuminate the tissue or can be ferroelectrically modulated. The ferroelectric modulation of the beam allows the generation of microsecond pulses of light. This modulation can be controlled by external hardware. This procedure not only dramatically reduces the temporal jittering of light pulses but also allows mixing beams of different wavelengths. The mixing of beams is done by multiplexing rays from different lasers. As a consequence, multiple dyes having different spectral properties can be excited to perform measurements of a variety of physiological variables, for example, Rhod-2 for cytosolic Ca2+, MagFluo4 for intra-SR Ca2+ and di-8-ANEPPS for membrane potential.
Although lasers present various advantages as the light source in LFFM, other types of light sources can be used including light-emitting diodes (LEDs). In this case, the excitation light source consisted of an InGaN LED (Figure 1c, PLEDFM). In LEDs, photons are spontaneously emitted when electrons from the conduction band recombine with holes in the valence band. The difference with solid-state lasers is that the emission is not stimulated by other photons. This results in a non-coherent beam and a wider spectral emission for LEDs.
Different types of high power LEDs can be used. For AP recordings using Di-8-ANEPPS and for Ca2+ transients recorded using Fluo-4 or Mag-Fluo-4, we used an LED that has a typical peak emission at 485 nm (blue) and a half width of 20 nm (Figure 1d). For Ca2+ transients recorded with Rhod-2, the LED had a typical peak emission at 540 nm (green) and a half width of 35 nm (Figure 1d). LEDs emit in a band wavelength and therefore require filters to narrow their spectral emission. In addition, pulsed light can be generated at a rate of 1.6 kHz with duration of 20 &#181;s. The LEDS were pulsed with a fast power MOSFET field effect transistor. Simultaneous recordings with different indicators can be performed by time-multiplexing the LEDs. Unfortunately, light emitted by LEDs is more difficult to focus onto a fiber optic compared to a laser beam. Thus, the main drawback of using LEDs is that their emission profiles have angular displacements (&#177; 15&#176;) from the main axis, and an auxiliary optic must be used to correct it.
In all of the optical configurations previously described, the excitation light is reflected with the aid of a dichroic mirror. The beam is subsequently focused by an aspheric lens and a microscope objective onto a multimode fiber optic that is positioned on the tissue. As in any epifluorescence arrangement, the dichroic mirror also serves to separate the excitation from the emitted light. The emitted light spectrum travels back through a barrier filter to remove any reflected excitation. Finally, the emitted light is focused with an objective onto a photodetector (Figure 1).
The transduction from light to electrical current is performed by silicon avalanche photodiodes. These diodes have a fast response and a high sensitivity allowing low light detection. The photocurrent produced by the avalanche photodiodes can be amplified in two ways: a transimpedance amplifier having a resistive feedback element (Figure 1e) or by an integrator to convert the current into a voltage (Figure 1f). Using the first approach, the output voltage is proportional to the photocurrent and the feedback resistor. A typical example of the resistive detection of picosecond laser pulses is shown in Figures 2a, 2b and 2c. Panel 2a illustrates the output of the transimpedance amplifier and panel 2b shows a time expansion of the interval indicated with an asterisk (*). A peak tracking algorithm was implemented to detect the peak (red) and the base (green) for the fluorescent responses12. The measurement of the base fluorescence provides information of both the dark current of the avalanche photodiode and the interferences introduced by ambient light and electromagnetic coupling. A representation of peaks and bases is shown in Figure 2c. This figure illustrates the fluorescence emitted by the dye (Rhod-2) bound to Ca2+ during the cardiac cycle of a beating parakeet heart.
In the second method, the output voltage of the integrator is a function of the current and capacitive feedback (Figures 2d, 2e, and 2f). Figure 2f shows two consecutive integration cycles: the first with no external illumination and the second with applied light pulses from a pulsed LED. A detailed description is presented in Figures 2g and 2h. This approach, although more laborious, provides a larger S/N due to the absence of thermal noise in the feedback capacitor. The instrument includes a timing stage that generates all the control and multiplexing of the excitation light and commands the headstage integration and reset periods. This is usually performed with a digital signal processing circuit that also performs a digital differentiation of the integrated output signal by computing an on-line regression of the data. In the case of using a resistive feedback, any A/D acquisition board can be used.
Finally, our LFFM technique is highly versatile and can be adapted to record from more than one region. Adding a beam splitter in the light path allows us to split the light into two optical fibers. Each optical fiber can then be placed on different regions of a tissue to, independently, excite and record emission from the exogenous fluorescent probes. This modification permits us to assess how anatomical regional differences influence physiological variables. Figure 3 shows a beam splitter being employed to split the CW excitation light such that two optical fibers are used to measure transmural electrical or intracellular [Ca2+] levels with minor-invasiveness. Transmural signals can be recorded by placing one fiber on the endocardium and the other on the epicardium layer of the ventricular wall. Therefore, the LFFM technique has the ability to measure the time course of cellular signals in different regions and can be used to test if regional changes occur under pathological situations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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local field fluorescence microscopy: imaging cellular signals in intact hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

AP and Ca2+ transients in endocardium and epicardium
In order to compare signals across the ventricular wall, one fiber optic is positioned in the endocardium and the other in the epicardium. Comparing the morphology of an AP recorded from the endocardium with one from the epicardium is the best way to assess the transmural function. The ventricular wall is highly hetero...

Watch this Scientific Journal Video about Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts at JoVE.com

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and ...

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.

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