Yoshihisa Yamaoka

Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region

The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.

Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy

Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.

Fine depth resolution of two-photon absorption-induced photoacoustic microscopy using low-frequency bandpass filtering

Photoacoustic microscopy usually uses high-frequency photoacoustic waves, which provide not only high spatial resolution but also limitation of the penetration depth. In this study, we developed two-photon absorption-induced photoacoustic microscopy (TP-PAM) to improve the depth resolution without use of high-frequency photoacoustic waves. The spatial resolution in TP-PAM is determined by two-photon absorption. TP-PAM with a 20X objective lens (numerical aperture: 0.4) provides an optically-determined depth resolution of 44.9 ± 2.0 μm, which is estimated by the full width at half maximum of the photoacoustic signal from an infinitely small target, using low-frequency bandpass filtering of photoacoustic waves. The combination of TP-PAM and frequency filtering provides high spatial resolution.

Low-energy laser irradiation promotes synovial fibroblast proliferation by modulating p15 subcellular localization.

Low‐energy laser irradiation (low‐level laser therapy) (LELI/LLLT/photobiomodulation) has been found to modulate various biological effects, especially those involved in promoting cell proliferation. Synovial fibroblasts are important in maintaining the homeostasis of articular joints and have strong chondrogenetic capacity. Here, we investigated the effect and molecular basis of LELI on synovial fibroblast proliferation.

Dual-wavelength excitation of mucosal autofluorescence for precise detection of diminutive colonic adenomas

BACKGROUND The mucosal layer of the colon contains metabolism-related fluorophores, such as reduced nicotinamide adenine dinucleotide (NADH), which might have the potential to serve as biomarkers for detecting neoplasia. OBJECTIVE To examine NADH fluorescence in human colonic adenoma while eliminating the effect of hemoglobin absorption and to develop a novel imaging technique for precise detection of adenomas. DESIGN Cross-sectional study. PATIENTS AND INTERVENTIONS A total of 66 endoscopically resected colonic polyps were investigated. After serial acquisition of autofluorescence images between 450 and 490 nm illuminated with dual-wavelength excitation at 365 nm (F(365ex)) and 405 nm (F(405ex)) on cross sections of the samples, ratio images were created by dividing F(365ex) by F(405ex). The excitation-emission wavelength combinations in F(365ex) and F(405ex) were optimized for NADH fluorescence and reference fluorescence. MAIN OUTCOME MEASUREMENTS The F(365ex)/F(405ex) ratio in the tumorous (T) and normal (N) mucosa. RESULTS F(365ex)/F(405ex) ratio images showed a 1.81- and 1.12-fold higher signal intensity in the adenomas and hyperplastic polyps, respectively, than in the adjacent normal mucosa. The ratio between signal intensities in tumorous mucosa and normal mucosa in F(365ex)/F(405ex) ratio images for tubular adenomas was significantly higher than that for hyperplastic polyps. The signal intensity in F(365ex)/F(405ex) ratio images was not correlated with the hemoglobin concentration index evaluated by reflection images at 550 nm and 610 nm. Diminutive adenomas (<5 mm) and large adenomas were well discriminated in F(365ex)/F(405ex) ratio images. LIMITATIONS Ex vivo experiment. CONCLUSIONS These results suggest that the precise measurement of NADH fluorescence intensity together with eliminating the influence of blood hemoglobin concentration serves as a method for visualizing colonic adenomas and that the dual-wavelength excitation method is a promising technique applicable to endoscopic detection of early colonic adenomas.

Intracellular dynamics of topoisomerase I inhibitor, CPT-11, by slit-scanning confocal Raman microscopy

Most molecular imaging technologies require exogenous probes and may have some influence on the intracellular dynamics of target molecules. In contrast, Raman scattering light measurement can identify biomolecules in their innate state without application of staining methods. Our aim was to analyze intracellular dynamics of topoisomerase I inhibitor, CPT-11, by using slit-scanning confocal Raman microscopy, which can take Raman images with high temporal and spatial resolution. We could acquire images of the intracellular distribution of CPT-11 and its metabolite SN-38 within several minutes without use of any exogenous tags. Change of subcellular drug localization after treatment could be assessed by Raman imaging. We also showed intracellular conversion from CPT-11 to SN-38 using Raman spectra. The study shows the feasibility of using slit-scanning confocal Raman microscopy for the non-labeling evaluation of the intracellular dynamics of CPT-11 with high temporal and spatial resolution. We conclude that Raman spectromicroscopic imaging is useful for pharmacokinetic studies of anticancer drugs in living cells.

Detection of Lymph Node Metastases in Human Colorectal Cancer by Using 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence with Spectral Unmixing

Accurate evaluation of metastatic lymph nodes (LNs) is indispensable for adequate treatment of colorectal cancer (CRC) patients. Here, we demonstrate detection of metastases of human CRC in removed fresh LNs using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence. A spectral unmixing method was employed to reduce the overlap of collagen autofluorescence on PpIX fluorescence. A total of 17 surgery patients with advanced CRC were included in this study. After 5-ALA at a dose of 15 mg/kg of body weight was applied orally 2 h prior to surgery, 87 LNs were subjected to spectral fluorescence imaging and histopathological diagnosis, and statistical analysis was performed. No apparent side effect was observed to be associated with 5-ALA administration. The spectral unmixing fluorescence intensity of PpIX in metastatic LNs was 10.2-fold greater than that in nonmetastaic LNs. The receiver-operating-characteristic (ROC) analysis showed that the area under the curve (AUC) was calculated as 0.95. Our results show the potential of 5-ALA-induced PpIX fluorescence processed by spectral unmixing for detecting metastases in excised fresh LNs from patients with CRC, suggesting that this rapid and feasible method is applicable to gross evaluation of resected LN samples in pathology laboratories.

Enhancement of multiphoton excitation-induced photoacoustic signals by using gold nanoparticles surrounded by fluorescent dyes

Recently, we have developed multiphoton excitation-induced photoacoustic imaging for thick tissues employing a 1064-nm nanosecond pulsed laser. The combination of multiphoton excitation and photoacoustic imaging improves the depth resolution. To apply the multiphoton-photoacoustic imaging for precise investigation in living tissues, it is important to enhance only the photoacoustic signals induced by multiphoton excitation, because the generation of multiphotonphotoacoustic signals is less efficient than that of one-photon photoacoustic signals. In this study, we investigated the relation between the signal intensity and the thermophysical properties of various solutions of fluorescent dyes in multiphoton-photoacoustic imaging. We found that the signal intensity is proportional to the coefficient of thermal expansion divided by the specific heat of the solvent. Thus thermophysical properties are also important, together with absorption properties, in enhancing the multiphoton-photoacoustic signal. Based on our findings, we propose the use of gold nanoparticles surrounded by fluorescent dyes as contrast agents. Rhodamine B, which is employed in fluorescent dyes, selectively evokes the two-photon absorption. In addition, because gold nanoparticles have a small specific heat, the multiphoton-photoacoustic signal generated is strong due to effective photon-to-heat conversion. We conclude that this combination allows deeper observation in living tissues by multiphoton-photoacoustic imaging.

Imaging of anticancer agent distribution by a slit-scanning Raman microscope

In recent years, various types of molecular imaging technologies have been developed, but many of them require probes and may have some influence on the distribution of the target molecules. In contrast, Raman microscopic analysis is effective for molecular identification of materials, and molecular imaging methods employing Raman scattering light can be applied to living organisms without use of any exogenous probes. Unfortunately, Raman microscopic imaging is rarely used in the biomedical field due to the weakness of Raman signals. When the conventional Raman microscopes are used, the acquisition of an image of a cell usually takes several hours. Recently, a slit-scanning confocal Raman microscope has been developed. It can acquire images of living cells and tissues with faster scanning speed. In this study, we used the slit-scanning confocal Raman microscope (RAMAN-11) to image the distribution of a drug in living cells. We could acquire images of the distribution of an anticancer reagent in living cells within several minutes. Since the wavelength of Raman scattering light is determined by the frequency of molecular vibration, the in situ mapping of the intracellular drugs without use of a probe is possible, suggesting that laser Raman imaging is a useful method for a variety of pharmacokinetic studies.

Photoacoustic microscopy using ultrashort pulses with two different pulse durations

We propose photoacoustic microscopy using ultrashort pulses with two different pulse durations in the range from femtoseconds to picoseconds. The subtraction of images for longer-pulse excitation from those for shorter-pulse excitation extracts two-photon photoacoustic images effectively, based on observation that the intensity ratio of two-photon to one-photon absorption-induced photoacoustic signals depends on the pulse duration in the same manner as the intensity ratio of two-photon and one-photon fluorescence signals. Two-photon photoacoustic microscopy using this subtraction method enables precise observation of the cross-sections of silicone hollows filled with the mixture of one-photon and two-photon absorption solutions.