Yimei Huang

Calcium mobilization in HeLa cells induced by nitric oxide.

Nitric oxide (NO) has been proposed to be involved in tumor growth and metastasis. However, the mechanism by which nitric oxide modulates cancer cell growth and metastasis on cellular and molecular level is still not fully understood. This work utilized confocal microscopy and fluorescence microplate reader to investigate the effects of exogenous NO on the mobilization of calcium, which is one of the regulators of cell migration, in HeLa cells. The results show that NO elevates calcium in concentration-dependent manner in HeLa cells. And the elevation of calcium induced by NO is due to calcium influx and calcium release from intracellular calcium stores. Moreover, calcium release from intracellular stores is dominant. Furthermore, calcium release from mitochondria is one of the modulation pathways of NO. These findings would contribute to recognizing the significance of NO in cancer cell proliferation and metastasis.

Evidence for A1 and A3 receptors mediating adenosine-induced intracellular calcium release in the dorsal root ganglion neurons by using confocal microscopy imaging

Adenosine exerts a key role in analgesia. In the present study, adenosine-induced Ca2+ responses were revealed by using confocal microscopy imaging in the rat dorsal root ganglia (DRG) neurons in vitro. Our results showed that adenosine could evoke increases in the intracellular Ca2+ concentration in the DRG neurons. In addition, by application of selective receptor antagonists, two types of receptors, A1R and A3R, were identified to be involved in the adenosine-induced Ca2+ release from intracellular stores in neurons. Altogether, these results suggest that confocal microscopy imaging combined with fluorescent dyes could help to detect the analgesic-induced ion signaling in single cell.

Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer

Abstract. Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors’ expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

CHARACTERIZATION OF SIGNAL CONDUCTION ALONG DEMYELINATED AXONS BY ACTION-POTENTIAL-ENCODED SECOND HARMONIC GENERATION

Action-potential-encoded optical second harmonic generation (SHG) has been recently proposed for use in detecting the axonal damage in patients with demyelinating diseases. In this study, the characterization of signal conduction along axons of two different levels of demyelination was studied via a modified Hodgkin–Huxley model, because some types of demyelinating disease, i.e., primary progressive and secondary progressive multiple sclerosis, are difficult to be distinguished by magnetic resonance imaging (MRI), we focused on the differences in signal conduction between two different demyelinated axons, such as the first-level demyelination and the second-level demyelination. The spatio-temporal distribution of action potentials along demyelinated axons and conduction properties including the refractory period and frequency encoding in these two patterns were investigated. The results showed that demyelination could induce the decrease both in the amplitude of action potentials and the ability of frequency coding. Furthermore, the signal conduction velocity in the second-level demyelination was about 21% slower than that in the first-level demyelination. The refractory period in the second-level demyelination was about 32% longer than the first-level. Thus, detecting the signal conduction in demyelinated axons by action-potential-encoded optical SHG could greatly improve the assessment of demyelinating disorders to classify the patients. This technique also offers a potential fast and noninvasive optical approach for monitoring membrane potential.

A porous model of human forearm for heat transfer analysis by finite element method

Heat transfer in biological tissues based on the theory of porous media has recently attracted attention because porous media play an important role in many biomedical applications. In this study, a porous model of human forearm was proposed, which took the blood-tissue convection into consideration. Finite element method was used to analyze the heat transfer equation. In addition, the influences of porosity, metabolic heat generation rate and environment temperature on the temperature distribution of human forearm were investigated. The simulation results showed that the temperature of the arterial vessel was higher than surrounding tissues. The temperature of muscle increased when the tissue porosity, metabolic heat generation rate and environment temperature increased. This study showed that the porous model could be a feasible and potential model for the study of human body thermal simulation.

Detection of oligomerization of Epstein-Barr virus oncoprotein LMP1 by FRET-based probes

In this study, two FRET-based probes are constructed to research oligomerization of Epstein-Barr virus Oncoprotein LMP1 in live cells. The images of wide-field fluorescence microscopy display that the majority of two LMP1-associated probes co-localized in internal perinuclear membranes. Furthermore, the fluorescence spectra of single cell co-expressed two probes indicated that the ratio of two emission peaks is around one, and the fluorescence spectra changed insignificantly during an hour observation. These findings indicated that LMP1/LMP1 interacted stably in live cells.

Combined tunable filters based swept laser source for optical coherence tomography

We demonstrate a novel ultra-broad tunable bandwidth and narrow instantaneous line-width swept laser source using combined tunable filters working at 1290 nm center wavelength for application in optical coherence tomography. The combined filters consist of a fiber Fabry-Perot tunable filter (FFP-TF) and a polygon mirror with scanning grating based filter. The FFP-TF has the narrow free spectral range (FSR) but ultra-high spectral resolution (narrow instantaneous bandwidth) driven at high frequency far from resonant frequency. The polygon filter in the Littrow configuration is composed of fiber collimator, polygon mirror driven by function generator, and diffractive grating with low groove. Polygon filter coarsely tunes with wide turning range and then FFP-TF finely tunes with narrow band-pass filtering. In contrast to traditional method using single tunable filter, the trade-off between bandwidth and instantaneous line-width is alleviated. The combined filters can realize ultra wide scan range and fairly narrow instantaneous bandwidth simultaneously. Two semiconductor optical amplifiers (SOA) in the parallel manner are used as the gain medium. The wide bandwidth could be obtained by these parallel SOAs to be suitable for sufficient wide range of the polygon filter’s FSR because each SOA generates its own spectrum independently. The proposed swept laser source provides an edge-to-edge scanning range of 180 nm covering 1220 to 1400 nm with instantaneous line-width of about 0.03 nm at sweeping rate of 23.3 kHz. The swept laser source with combined filters offers broadband tunable range with narrow instantaneous line-width, which especially benefits for high resolution and deep imaging depth optical frequency domain imaging.

Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

Examination of a demyelinated fiber by action-potential-encoded second harmonic generation

Axonal demyelination is a common phenomenon in the nervous system in human. Conventional measured approaches such as surface recording electrode and diffusion tensor imaging, are hard to fast and accurately determine the demyelinated status of a fiber. In this study, we first presented a mathematical model of nerve fiber demyelination, and it was combined with second harmonic generation(SHG) technique to study the characteristics of action-potential-encoded SHG and analyze the sensitivity of SHG signals responded to membrane potential. And then, we used this approach to fast examine the injured myelin sheaths resulted from demyelination. Each myelin sheath of a fiber was examined simultaneously by this approach. The results showed that fiber demyelination led to observable attenuation of action potential amplitude. The delay of action potential conduction would be markedly observed when the fiber demyelination was more than 80%. Furthermore, the normal and injured myelin sheaths of a myelinated fiber could be distinguished via the changes of SHG signals, which revealed the possibility of SHG technique in the examination of a demyelinated fiber. Our study shows that this approach may have potential application values in clinic.

Comparison of calcium imaging in dorsal root ganglion neurons by using laser scanning confocal and two-photon microscopy

As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.