Optical Fluorescence Imaging

Fluorescence imaging is widely used in cardiac research^{74, 98, 99}and was also extensively used in this thesis, see chapter 6. Fluorescence imaging allows the visualization of various physiological pro-cesses in biological cells and tissues and is very frequently applied to image neuronal and cardiac cells. In cardiac research, fluorescence imaging is commonly referred to asoptical mapping, eluding to its capability of mapping physiological activity also over larger surfaces of tissues at high spatial and temporal resolutions, enabling scientists to visualize, for instance, action potential wave patterns propagating over the epicardial surface of the ventricles. A review of the state-of-the-art of optical mapping techniques is given byHerron et al.²¹³

Fluorescence imaging employs wavelength-dependent light-tissue interactions. Visualization of in-tramural activity is therefore severely limited by the penetration depth of the light through tissue. As penetration depths of light in biological tissues typically range below<1mm, fluorescence imaging can not simply be performed transmurally⁴⁷through entire organs such as the heart muscle or in the presence of opaque materials like blood. The non-contact optical imaging modality offers

numer-Chapter 3. Imaging of Electromechanical Wave Activity in the Heart

dye excitation peak emission peak parameter sensitivity

Di-4-ANEPPS 488nm 605nm Vm

Di-8-ANEPPS 468nm 635nm Vm

Annine 6 450nm 515nm V_m

Di-4-ANBDQPQ 640nm 750nm Vm

Rhod-2AM 550nm 571nm [Ca²⁺]i

Fluo-3 480nm 535nm [Ca²⁺]_i

Fluo-4 494nm 516nm [Ca²⁺]i

Table 3.2:Fluorescent dyes commonly used in optical mapping experiments with cardiac tissue: voltage- (Vm) and calcium-sensitive ([Ca²⁺]i) dyes

ous advantages over the use of ECG electrodes, among which is the high spatial resolution and the prevention of electrophysiological artifacts and mechanical distortions introduced by the electrodes being inserted into the tissue. Fluorescence imaging was used for the visualization of cardiac arrhyt-mias such as ventricular or atrial tachycardia or fibrillation in intact, isolated whole hearts⁴⁴as well as in monolayer cardiac cell culture preparations.115, 136, 146, 154

3.1.1 Fluorescent Probes

Fluorescence imaging employs light to both illuminate and excite fluorescent probes introduced to the tissue. The probes can either be introduced to the intracellular space or may attach to the cell mem-brane and - once excited with light at the right wavelength - exhibit fluorescence^{18, 20}. Depending on physiological changes of the cell the fluorescence undergoes variations in its intensity or spectrum and these changes can be captured using optical filtering techniques and cameras. Currently, fluores-cent probes are most commonly used to report transmembrane potentialVmand intracellular Calcium concentration[Ca²⁺]_i, however, also magnesium, sodium, potassium, pH, nitric oxide, redox state and oxygen content can be reported.²¹³ The fluorescent probes can either be introduced to the tissue shortly before the measurement by staining the tissue preparation with dyes appropriately or can al-ready be present inside the tissue at the time of measurement in genetically modified tissues which express auto-fluorescence. Auto-fluorescence occurs for instance in transgenic cardiac tissue con-taining green fluorescent proteins (GFP) or cyan fluorescent proteins (CFP). Fluorescent dyes can be characterized by their parameter sensitivity, their excitation and emission bandwidths, their response times and fractional fluorescence intensity changes with respect to electrophysiological changes. The most commonly used dyes are styryl dyes, of which aminonaphthylethenylpyridnium (ANEP) dyes are the most prominent ones. Throughout this work, the dye di4-ANEPPS was used. A list of flu-orescent dyes together with their excitation- and absorption- and emission- peak wavelengths and the parameter sensitivity, that is the sensitivity to the membrane potential or the intracellular calcium concentration, is given in table 3.2. Each dye is commonly used in optical mapping experiments with cardiac tissue. In whole-heart optical mapping experiments, the tissue is stained by injecting fluorescent dye into the aorta from which it is distributed to the tissue via the vascular system, see chapter 6.

3.1.2 Imaging Hardware

The hardware used for fluorescence imaging consists of illumination, optical filtering equipment, lenses and typically high-speed cameras and acquisition hard- and software. High-speed cameras are

used to detect the fluorescence light. Most commonly used detectors are photodiode arrays (PDA), charged-couple device (CCD) and complementary metal oxide semiconductor (CMOS) cameras, each of which possess their own advantages and disadvantages. With the detector arrays it is possible to acquire simultaneously temporal electrophysiological signals from∼10.000sites with temporal resolutions of< 1ms. For instance, the SciMedia MiCAM ULTIMA (Brainvision, Japan) CMOS camera operates with a spatial resolution of100×100pixels at a temporal resolution of0.1−10kHz.

The Photometrics Cascade CCD camera operates with a spatial resolution of 128×128pixels at a temporal resolution of 0.1−0.5kHz. The optical setup of an optical mapping system consists of objectives with low numerical aperture to direct the excitation light onto and collect in the same time emitted fluorescence light from the tissue. Furthermore, a set of bandwidth barrier filters and dichroic filters is used to separate excitation light from emission light in the optical path of the optical map-ping system. Therefore, it is guaranteed that only fluorescence reaches the detector. Fluorescence imaging can be applied to single cells and groups of cells at cellular and subcellular levels as well as to whole populations of cells at the whole heart organ level. Here, the focus is set on the macroscopic approach with large fields of view ranging from 1×1mm² to3×3cm². Fluorescence imaging can be used in mono-parametric as well as in multi-parametric imaging mode, capturing multiple physiological parameters, such as transmembrane potential and intracellular calcium transients, si-multaneously. Fluorescence imaging can also be used in a panoramic fashion, imaging the cardiac cell culture preparations with multiple cameras from multiple directions.

3.1.3 Post-Procesing

Fluorescence imaging captures small fractional changes in fluorescence intensity that are immedi-ately caused by physiological activity of the tissue. Common techniques to visualize the small frac-tional changes in the fluorescence intensityI_f(t)are to normalize the temporal signal in each pixel over time or to subtract its mean over time from the signal itself, thus amplifying the intensity varia-tions. This can be done under the assumption that each pixel~p(x, t)continuously shows the same site of the tissue. Spatial gaussian, box car or cone filtering may be applied to improve signal-to-noise ratio. In case the tissue exhibits motion and the recorded frames show different tissue configura-tions χt the fluorescence variations of each site can not be associated with each pixel any longer, see section 6.4. The image contains motion artifacts. Then, the motion needs to be registered post-acquisition and each site exhibiting fluorescence needs to be tracked over space and time before the aforementioned analysis can be applied.

3.1.4 Clinical Applications

There are no clinical applications using conventional fluorescence imaging as the involved chemical compounds in fluorescent dyes are toxic. However, the field of optogenetics explores the possibility of creating transgenic tissue, with which, in principle, non-invasive fluorescence imaging should be possible.

Chapter 3. Imaging of Electromechanical Wave Activity in the Heart