To image the single molecule in vivo, we prepared very bright multiple quantum dots including several particles of quantum dots The solution of quantum dots was rapidly frozen in liquid nitrogen. The dots were aggregated in the frozen process. The number of quantum dots including in the multiple quantum dots were 10-100. The multiple quantum dots bound antibodies were bound to living cells. The bright fluorescence image was obtained. We developed new imaging methods to visualize molecules under noninvasive condition. We developed new imaging methods to visualize molecules under noninvasive condition. Tumor was successfully formed using several cells line. To image the molecules, specific antibodies to recognize these cells were labeled with fluorescence quantum dots and then injected to tail vein after the formation of tumor. We successfully performed real time observation of quantum dots within breast cancer cells and on its membrane under noninvasive condition.

In vivo nano-imaging have the potential to investigated molecular dynamics and mechanism in living mammals with nanometer accuracy. In this study, in the xenograft tumors, the mean velocity of EB1-comets suggests that the velocity of MT tips in vivo is affected by surrounding microenvironment of the cells by using high-speed (100fps) and high-resolution (~20 nm) confocal fluorescent microscope. Likewise, using the same imaging system, in living heart, we found that the working range of sarcomere length (1.90 and 1.68 μm in diastole and systole, respectively) existed on the shorter resting distribution side, and the left ventricular developed pressure was linearly correlated with the sarcomere length change between diastole and systole on the order of 100 nm.

1) In cardiac muscle, a change in sarcomere length (SL) by a mere ~100 nm causes a substantial change in contractility. To accurately analyze the motion of individual sarcomeres with nanometer precision, we developed an experimental system for simultaneous nano-scale analysis of single sarcomere dynamics and intracellular Ca changes via the expression of AcGFP in Z-disks in primary-cultured rat neonatal cardiomyocytes. 2) A rapid increase in temperature to >~38°C induced Ca-independent high-frequency (~10 Hz) sarcomeric auto-oscillations(HSOs) in rat neonatal cardiomyocytes. 3) We developed a high-speed high-resolution in vivo cardiac imaging system in mice. This system enabled three-dimensional analyses of sarcomere dynamics during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. 4) We demonstrated that the Frank-Starling mechanism of the heart was dependent on the “on-off” equilibrium of the thin filament state.

We use optical trapping to show the single molecule properties and the effect of load on the mechanochemical cycle of the motor domain of human dynein. The double-headed motor domain is responsible for producing a high force of ~6 pN with a predominant step size of 8 nm. An unbinding force measurement indicates that dynein-microtubule binding is weak for the ADP-vanadate state and strong for the nucleotide-free, AMPPNP and ADP states. The unbinding force was weaker when dynein was pulled toward the minus end of microtubule. Our results suggest that force plays an important role in the mechanochemical cycle of dynein to ensure the increasing of a probability for rear-head detachment with strain. We imaged the trafficking of PAR-1 carrying vesicles to analyze the movement of activated PAR-1 after internalization. By the triple-view method consisting of dual-focus fluorescence and phase contrast optics, we detected endocytosis quantum dots 3-dimensional with high special precision.

We developed high-resolution cardiac imaging systems both in cells and in vivo. First, by using quantum dots (QDs), we measured the length of a single sarcomere in isolated rat cardiomyocytes. QDs provided a quantitative measurement of sarcomere length. Second, we measured sarcomere length in AcGFP-expressing rat neonatal cardiomyocytes(precision, 3 nm). Neonatal myocytes exhibited spontaneous sarcomeric oscillations, and the waveform properties were indistinguishable from those obtained in electric field stimulation. Third, we conducted Ca imaging in the isolated mouse heart. Ca waves/transients became synchronized by electric stimulation. Forth, by using the adenovirus vector system, we developed an experimental system allowing for the real-time imaging of sarcomeric motions in ventricular myocytes in the anesthetized mouse (precision,20 nm). These molecular imaging technologies will be useful for the development of a novel diagnostic device for heart disease in future studies.

動物個体の機能を分子レベルで理解するために、マウスin vivo（個体内）の分子挙動をナノイメージングする装置を開発し、in vivoにおける生体運動の機構を解明します。そのために、複数の同色あるいは異色の量子ドットからなる粒子を合成し、この粒子に特定の分子を結合してマウス組織内に導入し、新規開発のin vivoイメージング装置にて分子の運動を観察し、生体運動の分子機構を統合的に解明します。