Confocal microscopy allows for optical sectioning of tissues, thus obviating the need for physical sectioning and subsequent registration to obtain a three-dimensional representation of tissue architecture. However, practicalities such as tissue opacity, light penetration, and detector sensitivity have usually limited the available depth of imaging to 200 microm. With the emergence of newer, more powerful systems, we attempted to push these limits to those dictated by the working distance of the objective. We used whole-mount immunohistochemical staining followed by clearing with benzyl alcohol-benzyl benzoate (BABB) to visualize three-dimensional myocardial architecture. Confocal imaging of entire chick embryonic hearts up to a depth of 1.5 mm with voxel dimensions of 3 microm was achieved with a 10x dry objective. For the purpose of screening for congenital heart defects, we used endocardial painting with fluorescently labeled poly-L-lysine and imaged BABB-cleared hearts with a 5x objective up to a depth of 2 mm. Two-photon imaging of whole-mount specimens stained with Hoechst nuclear dye produced clear images all the way through stage 29 hearts without significant signal attenuation. Thus, currently available systems allow confocal imaging of fixed samples to previously unattainable depths, the current limiting factors being objective working distance, antibody penetration, specimen autofluorescence, and incomplete clearing.

The specialized conduction tissue network mediates coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, then spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricle being activated from the apex toward the base and is thought to represent HPS function. The development of mature HPS function in embryogenesis follows significant phases of cardiac morphogenesis. Initially, cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases, ventricular activation in the looped heart still follows the direction of blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, maturing to apex-tobase pattern. The embryonic chick heart has been studied intensively by both electrophysiological and morphological techniques, and the morphology of its conduction system (which is similar to mammals) is well characterized. One interesting but seldom studied feature is the anterior septal branch (ASB), which came sharply to focus (together with the rest of the ventricular conduction system) in our birthdating studies. Using an optical mapping approach, we show that ASB serves to activate ventricular surface between stages 16 and 25, predating the functionality of the His bundle/bundle branches. Heart morphogenesis and conduction system formation are thus linked, and studying the abnormal activation patterns could further our understanding of pathogenesis of congenital heart disease.

The hypothesis that inner layers of contracting muscular tubes undergo greater strain than concentric outer layers was tested by numerical modeling and by confocal microscopy of strain within the wall of the early chick heart. We modeled the looped heart as a thin muscular shell surrounding an inner layer of sponge-like trabeculae by two methods: calculation within a two-dimensional three-variable lumped model and simulated expansion of a three-dimensional, four-layer mesh of finite elements. Analysis of both models, and correlative microscopy of chamber dimensions, sarcomere spacing, and membrane leaks, indicate a gradient of strain decreasing across the wall from highest strain along inner layers. Prediction of wall thickening during expansion was confirmed by ultrasonography of beating hearts. Degree of stretch determined by radial position may thus contribute to observed patterns of regional myocardial conditioning and slowed proliferation, as well as to the morphogenesis of ventricular trabeculae and conduction fascicles. Developmental Dynamics 238:1535-1546, 2009.

No abstract

Nanoparticle‐infused polymer composites have long been an area of interest due to the typically stabilizing effects they have on the mechanical behavior of the composites, as well as added functionalities such as reduced surface resistance. Newer applications of these nanocomposites include dispersing optoelectronically functional nanoparticles in polymer matrices, for example, including luminescent nanocrystals in rigid slabs for luminescent solar concentrating photovoltaics and electrochromic panels. Dispersion of luminescent nanocrystals in stretchable polymers adds novel possibilities for applications, such as wearable health monitoring systems, flexible communications devices, and more. To leverage the exciting combined properties that these nanocomposites promise, there is a pressing need to evaluate and understand the interconnections between the optical effects, mechanical behavior, and the properties of the constituents. Herein, the optical and mechanical behavior of brightly emissive silicon nanocrystal/polydimethylsiloxane (PDMS) nanocomposites are investigated as a function of nanocrystal concentration and nanocrystal surface functionality. Mechanically, incorporating the nanocrystals at higher concentration leads to a dramatic reduction in the mechanical properties of the nanocomposite. The results suggest that the nanocrystal inclusions interfere with polymer cross‐linking, changing the mechanical behavior of the host PDMS as compared to the control.