Shinpei Okawa

Improvement of image quality of time-domain diffuse optical tomography with lp sparsity regularization

An lp (0 < p ≤ 1) sparsity regularization is applied to time-domain diffuse optical tomography with a gradient-based nonlinear optimization scheme to improve the spatial resolution and the robustness to noise. The expression of the lp sparsity regularization is reformulated as a differentiable function of a parameter to avoid the difficulty in calculating its gradient in the optimization process. The regularization parameter is selected by the L-curve method. Numerical experiments show that the lp sparsity regularization improves the spatial resolution and recovers the difference in the absorption coefficients between two targets, although a target with a small absorption coefficient may disappear due to the strong effect of the lp sparsity regularization when the value of p is too small. The lp sparsity regularization with small p values strongly localizes the target, and the reconstructed region of the target becomes smaller as the value of p decreases. A phantom experiment validates the numerical simulations.

Blue laser irradiation generates intracellular reactive oxygen species in various types of cells.

UNLABELLED Abstract Objective: The purpose of this study was to measure intracellular reactive oxygen species (ROS) production after laser irradiation in various types of cells. BACKGROUND DATA ROS are considered to be the key secondary messengers produced by low-level laser therapy (LLLT). Although various mechanisms for the effects of LLLT have been proposed, and intracellular ROS were indicated as the one of the key factors, direct measurement of intracellular ROS of several types of cells after different wavelength lasers irradiation has not been reported. MATERIALS AND METHODS Various types of cells were used in this study: mouse preadipocytes (3T3-L1), prechondrocytes (ATDC5), myoblasts (C2C12), mesenchymal stromal cells (KUSA-A1), lung cancer cells (LLC), insulinoma cells (MIN6), fibroblasts (NIH-3T3), human cervix adenocarcinoma cells (HeLa), macrophages differentiated from lymphocytes (THP-1) after treatment with phorbol ester, and rat basophilic leukemia cells (RBL-2H3). Cells were irradiated with a blue laser (wavelength: 405 nm), a red laser (wavelength: 664 nm) or a near infrared laser (wavelength: 808 nm) at 100 mW/cm(2) for 60 or 120 sec. Intracellular ROS levels were measured by fluorometric assay using the intracellular ROS probe, CM-H2DCFDA in a flow cytometer. RESULTS After a blue laser irradiation, intracellular ROS levels were increased in all types of cells. In contrast, intracellular ROS generation was not observed after irradiation with a red laser or near-infrared laser. CONCLUSIONS Potential sources of intracellular ROS were excited by blue laser irradiation, resulting in ROS production within cells. Although the low-level intracellular ROS should be generated after a red or a near-infrared laser irradiation, the only high level intracellular ROS were detected by the ROS probe used in this study. As ROS are considered to be key secondary messengers, the specific functional regulation of cells by laser irradiation will be studied in a future study.

Microfluidic image cytometry for measuring number and sizes of biological cells flowing through a microchannel using the micro-PIV technique

Microfluidic image cytometry is developed and validated both theoretically and experimentally, which is a method for the simultaneous measurement of the number and sizes of particles flowing through a microchannel using image sequence analysis and micro particle image velocimetry technique. Theoretical considerations on image formation in this method predict the image profile of a known particle and suggest that corrections are required in particle size measurement in order to cancel the effects of both diffraction and out-of-focus location. A dilution series of 2 µm polystyrene particle suspensions were measured and compared with the results obtained by conventional Burker–Turk hemocytometry for validation of the particle counting. For the particle diameter measurements, the diameters of 2, 5, 10 and 20 µm particles were measured and compared with the official values of the manufacturer. The results of the number and sizes of the particles measured by the proposed method agreed well with the reference values. We hope that the proposed method will be applicable to the quantitative study of platelet aggregation in blood flow and become a powerful diagnostic tool in the future.

Low Reactive Level Laser Therapy for Mesenchymal Stromal Cells Therapies

Low reactive level laser therapy (LLLT) is mainly focused on the activation of intracellular or extracellular chromophore and the initiation of cellular signaling by using low power lasers. Over the past forty years, it was realized that the laser therapy had the potential to improve wound healing and reduce pain and inflammation. In recent years, the term LLLT has become widely recognized in the field of regenerative medicine. In this review, we will describe the mechanisms of action of LLLT at a cellular level and introduce the application to mesenchymal stem cells and mesenchymal stromal cells (MSCs) therapies. Finally, our recent research results that LLLT enhanced the MSCs differentiation to osteoblast will also be described.

3D Analysis of Intracortical Microvasculature During Chronic Hypoxia in Mouse Brains

The purpose of this study is to determine when and where the brain microvasculature changes its network in response to chronic hypoxia. To identify the hypoxia-induced structural adaptation, we longitudinally imaged cortical microvasculature at the same location within a mouse somatosensory cortex with two-photon microscopy repeatedly for up to 1 month during continuous exposure to hypoxia (either 8 or 10% oxygen conditions). The two-photon microscopy approach made it possible to track a 3D pathway from a cortical surface arteriole to a venule up to a depth of 0.8 mm from the cortical surface. The network pathway was then divided into individual vessel segments at the branches, and their diameters and lengths were measured. We observed 3-11 vessel segments between the penetrating arteriole and the emerging vein over the depths of 20-460 μm within the 3D reconstructed image (0.46 × 0.46 × 0.80 mm(3)). The average length of the individual capillaries (<7 μm in diameter) was 67 ± 46 μm, which was not influenced by hypoxia. In contrast, 1.4 ± 0.3 and 1.2 ± 0.2 fold increases of the capillary diameter were observed 1 week after exposure to 8 % and 10% hypoxia, respectively. At 3 weeks from the exposure, the capillary diameter reached 8.5 ± 1.9 and 6.7 ± 1.8 μm in 8% and 10 % hypoxic conditions, respectively, which accounted for the 1.8 ± 0.5 and 1.4 ± 0.3 fold increases relative to those of the prehypoxic condition. The vasodilation of penetrating arterioles (1.4 ± 0.2 and 1.2 ± 0.2 fold increases) and emerging veins (1.3 ± 0.2 and 1.3 ± 0.2 fold increases) showed relatively small diameter changes compared with the parenchymal capillaries. These findings indicate that parenchymal capillaries are the major site responding to the oxygen environment during chronic hypoxia.

Regulation of miRNA expression by low-level laser therapy (LLLT) and photodynamic therapy (PDT).

Applications of laser therapy, including low-level laser therapy (LLLT), phototherapy and photodynamic therapy (PDT), have been proven to be beneficial and relatively less invasive therapeutic modalities for numerous diseases and disease conditions. Using specific types of laser irradiation, specific cellular activities can be induced. Because multiple cellular signaling cascades are simultaneously activated in cells exposed to lasers, understanding the molecular responses within cells will aid in the development of laser therapies. In order to understand in detail the molecular mechanisms of LLLT and PDT-related responses, it will be useful to characterize the specific expression of miRNAs and proteins. Such analyses will provide an important source for new applications of laser therapy, as well as for the development of individualized treatments. Although several miRNAs should be up- or down-regulated upon stimulation by LLLT, phototherapy and PDT, very few published studies address the effect of laser therapy on miRNA expression. In this review, we focus on LLLT, phototherapy and PDT as representative laser therapies and discuss the effects of these therapies on miRNA expression.

Total light approach of time-domain fluorescence diffuse optical tomography

In this study, time-domain fluorescence diffuse optical tomography in biological tissue is numerically investigated using a total light approach. Total light is a summation of excitation light and zero-lifetime emission light divided by quantum yield. The zero-lifetime emission light is an emitted fluorescence light calculated by assuming that the fluorescence lifetime is zero. The zero-lifetime emission light is calculated by deconvolving the actually measured emission light with a lifetime function, an exponential function for fluorescence decay. The object for numerical simulation is a 2-D 10 mm-radius circle with the optical properties simulating biological tissues for near infrared light, and contains regions with fluorophore. The inverse problem of fluorescence diffuse optical tomography is solved using time-resolved simulated measurement data of the excitation and total lights for reconstructing the bsorption coefficient and fluorophore concentration simultaneously. The mean time of flight is used as the featured data-type extracted from the time-resolved data. The reconstructed images of fluorophore concentration show good quantitativeness and spatial reproducibility. By use of the total light approach, computation is performed much faster than the conventional ones.

Dermal carbonyl modification is related to the yellowish color change of photo-aged Japanese facial skin

BACKGROUND The photo-aged facial skin is characterized by various unique features such as dark spots, wrinkles, and sagging. Elderly people, particularly Asians, tend to show a yellowish skin color change with photo-aging. However, there has been no analytical study conducted on this unique skin color change of the aged facial skin. OBJECTIVE The purpose of the present study is to examine whether the carbonyl modification in the dermal protein is involved in the yellowish color change that occurs in the photo-aged skin. METHODS Normal skin samples excised from the face, abdomen and buttock of variously aged Japanese were separated into the epidermal and the dermal portions. These skin samples were histologically examined for carbonyl modification. Moreover, an in vitro constructed dermis model composed of a contracted collagen gel was treated with acrolein or 4-hydroxynonenal. All these samples were also studied colorimetrically. RESULTS The dermal samples obtained from the photo-aged facial skin exhibited an appearance of yellowish color, whereas neither the facial epidermis nor the dermis obtained from the abdomen or buttock showed such a yellowish discoloration. The upper layer of the dermis that revealed the yellowish color showed elastosis whose elastic fibers were found to colocalize with carbonyl protein as detected by a labeled hydrazide, as well as by an immunohistochemical examination using the antibody against acrolein adduct. Experimental induction of carbonyl modification in a dermis model in vitro by a long-term treatment with acrolein or 4-hydroxynonenal was found to show the appearance of the yellowish change which was also proven by an increase in b* value of colorimetry. It was more pronounced than that induced by glycation. CONCLUSION Our present results strongly suggest that carbonyl modification of the dermal protein is involved in the production of the yellowish color change that is noted in the photo-aged facial skin.

Responses of Cancer Cells Induced by Photodynamic Therapy

Photodynamic therapy (PDT) involves the administration of a photosensitizer, followed by local irradiation of tumor tissues using a laser of an appropriate wavelength to activate the photosensitizer. Since multiple cellular signaling cascades are concomitantly activated in cancer cells exposed to the photodynamic effect, understanding the responses of cancer cells to PDT will aid in the development of new interventions. This review describes the possible cell-death signaling pathways initiated by PDT. In addition, we describe our latest findings regarding the induction of expression of miRNAs specific to apoptosis in cancer cells and the induction of antitumor immunity following PDT against cancer cells. A more detailed understanding of the molecular mechanisms related to PDT will potentially improve long-term survival of PDT treated patients.

Optogenetics: Novel Tools for Controlling Mammalian Cell Functions with Light

In optogenetics, targeted illumination is used to control the functions of cells expressing exogenous light-activated proteins. Adoption of the optogenetic methods has expanded rapidly in recent years. In this review, we describe the photosensitive channel proteins involved in these methods, describe techniques for their targeting to neurons and other cell types both within and outside the nervous system, and discuss their applications in the field of neuroscience and beyond. We focus especially on the channelrhodopsin protein ChR2, the photosensitive protein most commonly employed in optogenetics. ChR2 has been used by many groups to control neuronal activity, both in vitro and in vivo, on short time scales and with exquisite anatomical precision. In addition, we describe more recently developed tools such as opsin/G protein-coupled receptor chimeric molecules and a light-activated transgene system. In addition, we discuss the potential significance of optogenetics in the development of clinical therapeutics. Although less than a decade old, optogenetics is already responsible for enormous progress in disparate fields, and its future is unquestionably bright.