Photothermolysis induced by femtosecond (fs) lasers may be a promising modality in dermatology because of its advantages of high precision due to multiphoton absorption and deeper penetration due to the use of near infrared wavelengths. Although multiphoton absorption nonlinear effects are capable of precision targeting, the femtosecond laser photothermolysis could still have effects beyond the targeted area if a sufficiently high dose of laser light is used. Such unintended effects could be minimized by real time monitoring photothermolysis during the treatment. Targeted photothermolytic treatment of ex vivo mouse skin dermis was performed with tightly focused fs laser beams. Images of reflectance confocal microscopy (RCM), second harmonic generation (SHG), and two-photon fluorescence (TPF) of the mouse skins were obtained with integrated multimodal microscopy before, during, and after the laser treatment. The RCM, SHG, and TPF signal intensities of the treatment areas changed after high power femtosecond laser irradiation. The intensities of the RCM and SHG signals decreased when the tissue was damaged, while the intensity of the TPF signal increased when the photothermolysis was achieved. Moreover, the TPF signal was more susceptible to the degree of the photothermolysis than the RCM and SHG signals. The results suggested that multimodal microscopy is a potentially useful tool to monitor and assess the femtosecond laser treatment of the skin to achieve microscopic photothermolysis with high precision.
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.
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.
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.
Nitric oxide (NO) has been reported to have the ability to promote or inhibit the proliferation and metastasis of cancer
cells. It appears to have an effect on inducing calcium transient, which participates in essential cellular signaling in the
physiological and pathological processes. Our work was intended to study the effects of exogenous NO on intracellular
calcium dynamics of HeLa cells with Fluo-3, a calcium fluorescent indicator by microplate fluorescence reader. The
results showed that after NO donor was injected into the wells, intracellular Ca2+ fluorescence intensity increased
significantly compared with that of control group. Furthermore, the calcium transient activated by NO was mainly due to
the calcium release from intracellular calcium stores. These would be helpful to further recognize the role of NO
involved in cancer cell proliferation and metastasis.
Demyelination of a nerve fiber was simulated by action potential encoded second harmonic generation (SHG). The
dynamics of action potential propagation along a nerve fiber with a multi-internode demyelination happening to
successive internodes or intermittent internodes was studied. The results showed the attenuation and delay of action
potential could obviously occur, and the refractory period increased when a nerve was demyelinated. In addition, under
the same thickness and number of the demyelination, the peak of SHG signals attenuated much more along successive
injured internodes than along intermittent injured internodes. It indicated that action potential encoded SHG could be a
useful tool for detecting nerve demyelination.
Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study
was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root
ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine
on DRG neurons. After adding adenosine with different concentration, we compared the metabolic
changes by the real time imaging of calcium and mitochondria membrane potential using confocal
microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of
DRG neurons was more significant than others. Furthermore, four different adenosine receptor
antagonists were used to study which receptor mediated the influences of adenosine on the cultured
DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had
effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above
studies demonstrated that the effect of adenosine which may be involved in the signal transmission
on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R
may mediate the transmission.
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.
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.
The physical properties of acupuncture point were important to discover the mechanism of acupuncture meridian. In this
paper, we used an optical coherence tomography to monitor in vivo the changes of optical attenuation coefficients of
Hegu acupuncture point and non-acupuncture point during laser irradiation on Yangxi acupuncture point. The optical
attenuation coefficients of Hegu acupuncture point and non-acupuncture point were obtained by fitting the raw data
according to the Beer-Lambert's law. The experimental results showed that the optical attenuation coefficient of Hegu
acupuncture point decreased during the laser acupuncture, in contrast to a barely changed result in that of
non-acupuncture point. The significant change of optical attenuation coefficient of Hegu acupuncture point indicated that
there was a correlation between Hegu and Yangxi acupuncture points to some extent.
Nonablative skin remodeling is a new light treatment approach for photodamaged skin. Compared to ablative CO2 or
Er:YAG laser resurfacing, dermabrasion, and chemical peels, the clinical objective of nonablative skin remodeling is to
maximize thermal damage to upper dermis while minimizing injury to the epidermis and surrounding tissue,
consequently decreasing potential complications and shortening long recuperation periods.
Histological analysis of preoperative and postoperative biopsies using H&E or special stains has indicated the dermal
thermal injury, which resulting in collagen denaturation, is the most important mechanism of nonablative skin
remodeling for improving skin situation. And the extent of improvement of skin situation corresponded to the formation
of a new band of dense, compact collagen bundles in the papillary dermis. The diversity of individual skin condition
influences the choice of pulsed light treatment parameters, and further influences the degree of dermal thermal damage,
thus the efficacy of nonablative skin remodeling remains unstable.
Recently, multiphoton microscopy has show a promising application for monitoring skin thermal damage, because
collagen could produce strong second harmonic generation (SHG). And SHG intensity is presumably proportional to the
percentage of collagen in dermis. In this paper, the auto-fluorescence (AF) intensity and SHG intensity of mice skin
irradiated by pulsed Nd:YAG laser were measured and imaged with multiphoton microscope, and the results show the
ratio of SHG to AF decreases with the increase of irradiation exposure dose, and could be a quantitative technique to
assess dermal thermal damage, and could further benefit the choice of light treatment parameters.
With low risk of complications and no down-time, the non-ablative photorejuvenation is playing an increasing role
in the therapy of the photodamaged skin. The light dose is one of the key factors that affect the performance of the
photorejuvenation. Monitoring the tissue response during the procedure of laser irradiation would help to determine
whether the light dose is appropriate. In this study, we developed a new approach to monitor the instant response of
tissue irradiated by laser device by measuring the change of the total attenuation coefficient of the tissue with optical
coherence tomography. The total attenuation coefficient was deduced from the raw data obtained by OCT with single
scattering mode. Spatial and temporal equalizations were employed to improve the signal-to-noise ratio. We measured in
vivo the total attenuation coefficients of the mouse back skin before and after laser irradiation. The total attenuation
coefficients of the tissue reduced approximately 60% immediately after laser irradiation and then kept constant in a short
time. The reduction of the attenuation coefficient depended on the light dose. These results demonstrated that the new
approach could be a potential tool for monitoring in clinic in the future.
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