Single fluorescent dye molecules were visualized at video-rate in aqueous solution by using total internal reflection fluorescence microscopy. This approach enabled us to directly image single myosin molecules and detect individual ATP turnover reactions. This approach also can be used to directly image single fluorescently-labeled kinesin molecules moving on an axoneme. The methods can be extended to simultaneous measurements of the ATPase reaction and force/movement by single motor protein molecules. This should provide a clear answer to the fundamental problem of how mechanical reaction is coupled to ATPase reaction.
We have directly imaged organic molecules such as fatty acids and cholesterol adsorbed onto a graphite substrate using a scanning tunneling microscopy (STM) . The STM images have attained atomic resolution and as a result, we could distinguish the individual hydrocarbon groups of fatty acids. Moreover, two successive STM images reveal the molecular motion at the interface between a solution and the graphite substrate. Finally, we stress that STM imaging at the liquid/graphite interface should be a powerful technique for the observation of other organic molecules.
We sumrnarize the mechanical and functional properties of a motormolecule of muscle studied with a new type of an in vitro motility assay system and a multi-imaging microscope system. First, supercoiling of an actin filament is shown, suggesting that the filament moves forward like a right-handed screw. Second, we describe the unbinding force and the lifetime of a rigor bond between a single myosin molecule and an actin filament measured by using optical tweezers. From these results, the mechanism of sliding and force generation of the motor molecule is discussed.
Every element of life, at all levels of hierarchy down to a single molecule, is a functional entity that by itself performs a delicate task (s). Understanding of the mechanism of biological machinery therefore requires a close observation of the performance of individual cells, organelles, or molecules. Three examples are presented, in which single-cell or single-molecule observation under an optical microscope was crucial in the analysis: ionic regulation of the acrosome reaction in starfish sperm revealed by multi-view microscopy, elucidation of elementary steps of actomyosin motors using optical tweezers, and detection of molecular rotation via single fluorophore imaging.
Optical imaging with high spatial and temporal resolution of neural activity in rat cortical slices was used to investigate the dynamics of signal transmission through neural connections in the visual cortex. When inhibition due to γ-aminobutyric acid was slightly suppressed, horizontal propagation of excitation in both the supra- and infragranular layers became prominent. This propagation was not affected by vertical cuts in either the supra- or infragranular layer, which suggests that excitation is at least partially conveyed horizontally by reciprocal vertical connections between neurons in these layers.
Already more than 10 years have passed since the first publication of fluorescent Ca2+ indicator. The method of intracellular Ca2+ concentration measurement now became one of the standard experimental techniques in the filed of biosciences. The pseudo-color visualization of the Ca2+ dynamics in the living cell was quite convincing. This can be achieved by the computers and its related techniques which have tremendously developed in those 10 years. In this technical review we will overview the recent been development of fluorescent Ca2+ indicators and the techniques for image analysis of intracellular Ca2+ concentration.
Dynamic images of blood and lymph flow in the microcirculation can be obtained in-situ under the microscopic observation using a high speed video recording system. Red blood cell velocity and lymphocyte velocity were measured by the image correlation method and the position matching method respectively with a PC-based image processing system. Image analysis of the flow dynamics is highly effective to elucidate the organic homeostasis on the physiological and pathological points of view.
We developed a high resolution and high speed CCD intravital videomicroscope. The microscope system consists of a needle probe, camera body containing a CCD camera that has a high definition CCD image sensor and a high speed CCD sensor. The temporal resolution is 33 or 5 ms and the spatial resolution is 2.5 or 3.9μm. We could clearly, continuously observe diameters of botb subepicardial and subendocardial arterioles and venules throughout a cardiac cycle in a beating dog heart. The vascular compression by cardiac contraction decreased the diameters of both subendocardial arterioles and venules by about 20%, whereas subepicardial arterial diameter changed very little during the cardiac cycle and subepicardial venules increased in diameter during systole. The blood flow in the microvessels were successfully visualized by the high speed system with a bright microsphere. In conclusion, a newly developed microscope system is a powerful tool for observation of dynamic diameter and flow changes in coronary microvessels in a beating dog heart.