Bruce Zhi Gao

Within the myocardium, cardiomyocytes reside in a complex and dynamic extracellular matrix (ECM) consisting of a basement membrane (BM) and interstitial matrix. The interactions between cardiomyocytes and the myocardial ECM play a critical role in maintaining cardiac geometry and function throughout cardiac development and in adult hearts. Understanding how the structural changes of the myocardial ECM affect cardiomyocyte function requires knowledge of pericellular structures. These structures are of a size beyond the resolution of conventional optical microscopy. Here, we demonstrated multi-scale and multi-aspect characterization of the cardiomyocyte microenvironment in myocardial tissue sections using multimodal stimulated emission depletion (STED) microscopy. Second harmonic generation and autofluorescence facilitated multiplexed imaging, enabling the interpretation of protein distribution in 3D. STED imaging modality revealed BM structures of cardiomyocytes and myocardial capillaries at the subdiffractional level. Moreover, meaningful measurements retrieved from acquired images, such as sarcomere length and capillary density, enabled quantitative assessment of myocardial structures.

Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.

The super-resolution imaging technique of structured illumination microscopy (SIM) enables the mixing of high-frequency information into the optical transmission domain via light-source modulation, thus breaking the optical diffraction limit. Correlative SIM, which combines other techniques with SIM, offers more versatility or higher imaging resolution than traditional SIM. In this review, we first briefly introduce the imaging mechanism and development trends of conventional SIM. Then, the principles and recent developments of correlative SIM techniques are reviewed. Finally, the future development directions of SIM and its correlative microscopies are presented.

In vitro cultures to be used in various analytical investigations of cardiomyocyte growth and function for enhancing insight into physiological and pathological mechanisms should closely express in vivo morphology. The aim of the studies is to explore how to use microfabrication and physical-cue-addition techniques to establish a neonatal rat cardiomyocyte culture model that expresses an end-to-end connected rod shape with in vivo-like intercalated discs (ICDs). Freshly isolated neonatal rat cardiomyocytes were cultured on microgrooved PDMS (polydimethylsiloxane) substrate. Cell alignment and ICD orientation were evaluated using confocal fluorescence and transmission electron microscopy under various combinations of different culture conditions. Cyclic stretch and blebbistatin tests were conducted to explore mechanical and electrical effects. Lab-made Matlab software was developed to quantify cell alignment and ICD orientation. Our results demonstrate that the mechanical effect associated with the electrical stimulation may contribute to step-like ICD formation viewed from the top. In addition, our study reveals that a suspended elastic substrate that was slack with scattered folds, not taut, enabled cardiomyocyte contraction of equal strength on both apical and basal cell surfaces, allowing the cultured cardiomyocytes to express a 3D rod shape with disc-like ICDs viewed cross-sectionally.

Laser cell patterning is a distinctly effective technique for creating cell arrangements in culture that replicate in vivo tissue structure for studying contact-mediated cell-cell interactions. Conventional laser-based single-cell-manipulation techniques are limited by their inability to pattern irregularly shaped cells, such as rod-shaped cardiomyocytes. We report use of a spatial light modulator loaded with a computer-generated phase map to shape a single laser source into multiple laser-guidance beams distributed around the outer contour of an irregularly shaped cell to achieve accurate cell patterning. In addition to describing the principle and practice of the system design, we present what is to our knowledge the first achievement of patterning large, irregularly (rod) shaped adult rat cardiomyocytes in an end-to-end connected alignment to replicate the in vivo heart muscle cell connection without use of substrate surface modifications, which can interfere with in vivo-like cell-cell and cell-extracellular matrix interactions. Our research demonstrates that two-stage multiple beam laser guidance is effective: 1) A dual-beam configuration horizontally translates a cell from the outlet of the microfluidic cell delivery channel to a position above the cell deposition site and 2) A quad-beam configuration rapidly propels the cell axially through the suspension medium to the culture substrate with vertical movement of the cell patterning chamber. Our study reveals that 90% of the patterned cells maintained end-to-end connection 30 min after patterning, and mechanical junctions could be reinstalled between laser connected cells after overnight incubation. This demonstrates that multiple beam laser patterning is an outstanding tool for in vitro studying contact-mediated cell-cell interactions among irregularly shaped cells.

A growing number of research papers report similarities in cell types, neuronal connections and information‐processing principles between chick and rodent cortical tissues, which have very different architectures. This paper extends these comparisons beyond the cortical tissues. Using microelectrode array technology, we report three remarkable functional similarities between our original chick data and rodent data from the literature: (a) the pattern of spontaneous spiking activity from chick spinal cord neuron biosensors is very similar to that of rodent spinal cord neuron biosensors (i.e. rodent counterparts). (b) The spontaneous spiking activity pattern of the chick forebrain neuron biosensors is very similar to that of the rodent cortical neuron biosensors, but chick forebrain neuron biosensors contain not only cortical neurons, but also diencephalic neurons. In other words, chick forebrain neuron biosensors cannot be considered the counterparts of rodent cortical neuron biosensors. (c) Chick forebrain neuron biosensors respond to several classical neuroactive agents in a way similar to rodent cortical neuron biosensors as reflected in their agent‐specific concentration–response curves and their values of EC₅₀ (the effective concentration that causes 50% of the maximal effect of an agent). These preliminary findings provide both direct and indirect support for a positive answer to the big research question in the title: ‘Do chick and rodent neuron biosensors function similarly’ if the sources of the neurons are homologous between chick and rodent? Our findings are of particular value to comparative biology/physiology, pharmacology, neurotoxicology and bioengineering and to research on the more cost‐effective extended application of chick neuron biosensors.

Detecting the structural changes caused by volume and pressure overload is critical to comprehending the mechanisms of physiologic and pathologic hypertrophy. This study explores the structural changes at the crystallographic level in myosin filaments in volume- and pressure-overloaded myocardia through polarization-dependent second harmonic generation microscopy. Here, for the first time, we report that the ratio of nonlinear susceptibility tensor components d₃₃/d₁₅ increased significantly in volume- and pressure-overloaded myocardial tissues compared with the ratio in normal mouse myocardial tissues. Through cell stretch experiments, we demonstrated that mechanical tension plays an important role in the increase of d₃₃/d₁₅ in volume- and pressure-overloaded myocardial tissues.