Yonghong Shao

Myosin filament assembly onto myofibrils in live neonatal cardiomyocytes observed by TPEF-SHG microscopy

AIMS Understanding myofibrillogenesis is essential for elucidating heart muscle formation, development, and remodelling in response to physiological stimulation. Here, we report the dynamic assembly process of contractile myosin filaments onto myofibrils in a live cardiomyocyte culture during myofibrillogenesis. METHODS AND RESULTS Utilizing a custom-built, two-photon excitation fluorescence and second harmonic generation imaging system equipped with an on-stage incubator, we observed new sarcomere additions in rat neonatal cardiomyocytes during 10 h of on-stage incubation. The new sarcomere additions occurred at the side of existing myofibrils, where we observed mature myofibrils acting as templates, or at the interstice of several separated myofibrils. CONCLUSIONS During sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric alignment during maturation.

RECENT PROGRESS IN MULTIFOCAL MULTIPHOTON MICROSCOPY.

Multifocal multiphoton microscopy (MMM) has recently become an important tool in biomedicine for performing three-dimensional fast fluorescence imaging. Using various beamsplitting techniques, MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then records fluorescence signal from all foci simultaneously with an area array detector, which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light. In this paper, we discuss the features of several MMM setups using different beamsplitting devices, including a Nipkow spinning disk, a microlens array, a set of beamsplitting mirrors, or a diffractive optical element (DOE). In particular, we present our recent work on the development of an MMM using a spatial light modulator (SLM).

Micro-Raman spectroscopy study of cancerous and normal nasopharyngeal tissues

Abstract. The capabilities of micro-Raman spectroscopy for differentiating normal and malignant nasopharyngeal tissues were evaluated. Raman scattering signals were acquired from 22 normal and 52 malignant nasopharyngeal tissue samples. Distinctive spectral differences in Raman spectra between normal and malignant nasopharyngeal tissues were found, particularly in the spectral ranges of 853, 937, 1094, 1209, 1268, 1290 to 1340, 1579, and 1660  cm−1, which primarily contain signals related to proteins, DNA, and lipids. Compared to normal tissues, the band intensity located at 853, and 937  cm−1 were significantly lower for cancerous tissues (p<0.05), while the band intensity located at 1094, 1209, 1268, and 1579  cm−1 were significantly higher (p<0.05). The band intensity located at 1290 to 1340, and 1660  cm−1 were also higher for cancerous tissues; but the differences were not statistically significant (p>0.05). Principal component analysis (PCA) and linear discriminate analysis (LDA) were employed to generate diagnostic algorithms for classification of Raman spectra of the two nasopharyngeal tissue types. The PCA-LDA algorithms together with leave-one-out, cross-validation technique yielded diagnostic sensitivity of 92% and specificity of 82%. This work demonstrated that the Raman spectroscopy technique associated with PCA-LDA diagnostic algorithms has potential for improving the diagnosis of nasopharyngeal cancers.

Ultrafast, large-field multiphoton microscopy based on an acousto-optic deflector and a spatial light modulator

We present an ultrafast, large-field multiphoton excitation fluorescence microscope with high lateral and axial resolutions based on a two-dimensional (2-D) acousto-optical deflector (AOD) scanner and spatial light modulator (SLM). When a phase-only SLM is used to shape the near-infrared light from a mode-locked titanium:sapphire laser into a multifocus array including the 0-order beam, a 136 μm × 136 μm field of view is achieved with a 60× objective using a 2-D AOD scanner without any mechanical scan element. The two-photon fluorescence image of a neuronal network that was obtained using this system demonstrates that our microscopy permits observation of dynamic biological events in a large field with high-temporal and -spatial resolution.

Myofibrillogenesis in live neonatal cardiomyocytes observed with hybrid two-photon excitation fluorescence-second harmonic generation microscopy

We developed a hybrid two-photon excitation fluorescence-second harmonic generation (TPEF-SHG) imaging system with an on-stage incubator for long-term live-cell imaging. Using the imaging system, we observed the addition of new sarcomeres during myofibrillogenesis while a cardiomyocyte was spreading on the substrate. The results suggest that the TPEF-SHG imaging system with an on-stage incubator is an effective tool for investigation of dynamic myofibrillogenesis.

Addressable multiregional and multifocal multiphoton microscopy based on a spatial light modulator

Through a combination of a deflective phase-only diffractive spatial light modulator (SLM) and galvo scanners, an addressable multiregional and multifocal multiphoton microscope (AM-MMM) is developed. The SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple beamlet arrays with addressable shapes and sizes that match the regions of interest on the sample. Compared with conventional multifocal multiphoton microscope (MMM), AM-MMM achieves the effective use of the laser power with an increase of imaging rate and a decrease of photodamage without sacrifice of resolution.

3D myofibril imaging in live cardiomyocytes via hybrid SHG-TPEF microscopy

We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG and TPEF in the research of myofibrillogensis.

Fast flexible multiphoton fluorescence lifetime imaging using acousto-optic deflector

We present a fast and flexible fluorescence lifetime imaging microscopy which uses a two-dimensional acousto-optic deflector to achieve fast beam scanning across the sample and provides random access to the regions of interests (ROI). Experimental results using standard fluorescent dye and biological samples show that this system can make addressable fluorescence lifetime measurements and perform fast and flexible fluorescence lifetime imaging particularly to the discontinuous ROI in the sample.

Dynamic Myofibrillar Remodeling in Live Cardiomyocytes under Static Stretch

An increase in mechanical load in the heart causes cardiac hypertrophy, either physiologically (heart development, exercise and pregnancy) or pathologically (high blood pressure and heart-valve regurgitation). Understanding cardiac hypertrophy is critical to comprehending the mechanisms of heart development and treatment of heart disease. However, the major molecular event that occurs during physiological or pathological hypertrophy is the dynamic process of sarcomeric addition, and it has not been observed. In this study, a custom-built second harmonic generation (SHG) confocal microscope was used to study dynamic sarcomeric addition in single neonatal CMs in a 3D culture system under acute, uniaxial, static, sustained stretch. Here we report, for the first time, live-cell observations of various modes of dynamic sarcomeric addition (and how these real-time images compare to static images from hypertrophic hearts reported in the literature): 1) Insertion in the mid-region or addition at the end of a myofibril; 2) Sequential addition with an existing myofibril as a template; and 3) Longitudinal splitting of an existing myofibril. The 3D cell culture system developed on a deformable substrate affixed to a stretcher and the SHG live-cell imaging technique are unique tools for real-time analysis of cultured models of hypertrophy.

High-throughput imaging surface plasmon resonance biosensing based on an adaptive spectral-dip tracking scheme.

Imaging-based spectral surface plasmon resonance (λSPR) biosensing is predominantly limited by data throughput because of the multiplied data capacity emerging from 2-dimensional sensor array sites and the many data points required to produce an accurate measurement of the absorption dip. Here we present an adaptive feedback approach to address the data throughput issue in λSPR biosensing. A feedback loop constantly tracks the dip location while target-molecule binding occurs at the sensor surface. An adaptive window is then imposed to reduce the number of data points that each pixel has to capture without compromising measurement accuracy. Rapid wavelength scanning is performed with a liquid crystal tunable filter (LCTF). With the use of a feedback loop, our demonstration system can produce a dip measurement within 700ms, thus confirming that the reported λSPR approach is most suitable for real-time micro-array label-free biosensing applications.