Sayoko Yokoi

Ultra-high-speed bionanoscope for cell and microbe imaging

We are developing an ultra-high-sensitivity and ultra-high-speed imaging system for bioscience, mainly for imaging of microbes with visible light and cells with fluorescence emission. Scarcity of photons is the most serious problem in applications of high-speed imaging to the scientific field. To overcome the problem, the system integrates new technologies consisting of (1) an ultra-high-speed video camera with sub-ten-photon sensitivity with the frame rate of more than 1 mega frames per second, (2) a microscope with highly efficient use of light applicable to various unstained and fluorescence cell observations, and (3) very powerful long-pulse-strobe Xenon lights and lasers for microscopes. Various auxiliary technologies to support utilization of the system are also being developed. One example of them is an efficient video trigger system, which detects a weak signal of a sudden change in a frame under ultra-high-speed imaging by canceling high-frequency fluctuation of illumination light. This paper outlines the system with its preliminary evaluation results.

Fiber formation of a synthetic spider peptide derived from Nephila clavata

Dragline silk is a high‐performance biopolymer with exceptional mechanical properties. Artificial spider dragline silk is currently prepared by a recombinant technique or chemical synthesis. However, the recombinant process is costly and large‐sized synthetic peptides are needed for fiber formation. In addition, the silk fibers that are produced are much weaker than a fiber derived from a native spider. In this study, a small peptide was chemically synthesized and examined for its ability to participate in fiber formation. A short synthetic peptide derived from Nephila clavata was prepared by a solid‐phase peptide method, based on a prediction using the hydrophobic parameter of each individual amino acid residue. After purification of the spider peptide, fiber formation was examined under several conditions. Fiber formation proceeded in the acidic pH range, and larger fibers were produced when organic solvents such as trifluoroethanol and acetonitrile were used at an acidic pH. Circular dichroism measurements of the spider peptide indicate that the peptide has a β‐sheet structure and that the formation of a β‐sheet structure is required for the spider peptide to undergo fiber formation.

Phototriggering system for an ultrahigh-speed video microscopy

A phototrigger system is developed as a part of a video microscope mounting an ultrahigh-speed video camera capable of image capturing at frame rates as high as 1x10(6) framess. The extremely high frame rate is achieved by implementing in situ image storage. A distinguished feature of the camera is the on-chip overwriting mechanism that allows to keep in storage the latest image sequence of 103 frames; the old signals are continuously drained out of the storage. The trigger system is designed to synchronize recording operations with an occurrence of a target event within the limited image capturing duration. The target event is detected through a sudden change in the output of a sensor mounted to an optical port of the microscope. To reduce noise contribution, a two-sensor architecture is implemented. One sensor detects the target event while the one produces a reference signal used for noise reduction. Both sensors are connected to the same optical port by using a specially designed beam splitting unit. To provide high sensitivity, avalanche photodiodes are used as photoelements. System evaluation shows that its sensitivity is high and response time is less than 3 mus. This is sufficiently fast for high-speed video-microscopy observations at 1x10(6) frames/s when using a video camera with a storage of 103 frames. As an example, the system was used in a microscopic observation of a soap film collapse.

A bright and long-pulse illumination for ultrahigh-speed microscopy of living specimens

Ultrahigh-speed microscopy of living specimens requires ultrabright illumination. Moreover, the duration of illumination should be sufficiently long, on the order of at least several tens of milliseconds, in order to investigate the dynamic state of living specimens. However, specimens are exposed to a high risk of damage by the intense illumination. The brightness and pulse duration of illumination have to be continuously controlled for use in the ultrahigh-speed microscopy of living specimens. Commercial or laboratory-made illumination systems do not satisfy the abovementioned requirements. In this paper, the development of a bright and long-pulse illumination system for ultrahigh-speed microscopy of living specimens is presented. A xenon flashlamp with an arc length of 1.5 mm has been used as the light source. The electrical power supply consists of a voltage-regulated circuit, a capacitor bank, and a control circuit including an insulated-gate bipolar transistor as a gating device, which provides a large rectangular current pulse with the duration in the range to the order of several tens of milliseconds. The brightness, pulse duration, and repetition rate can be easily and continuously controlled. The illumination developed in the present study is installed in an inverted fluorescence microscope equipped with a high-speed camera in order to evaluate the performance as an illumination source. A fluorescent image of the living spermatozoa of a mouse obtained at a frame rate of 8 kHz shows good contrast. Such an image cannot be obtained using a commercial illumination system.

In search of techniques to obtain dynamic fluorescence images of cellular phenomena occurring at ultra-high speed

Although we have aspired to observe dynamic changes in fluorescent images at the cellular level for a long time, the commercially available video cameras are not at all suitable for this purpose because of their low frame rates and photosensitivity. The present work tackles this issue and describes our attempt to find a solution by using our high-speed video camera and an ultrabright illumination system. We used light sources with considerably higher energy because conventional mercury lamps cannot produce sufficient brightness for our video cameras working a rate of more than 4,500 fps to obtain fluorescent images of cells. We observed that the flagellar movement of mice sperms ceased and multiple kinks developed in their tails when exposed to 2.7W of laser illumination for 1 s. In contrast, no significant alterations could be detected when the sperms were subjected to the same amount of energy by intermittent illumination. Since we found that cells can survive short-duration exposure to high-energy light, we attempted to construct an ultrabright Xenon-strobe illumination system. Our fluorescence studies are currently being extended to other types of animal cells, e.g., observation of the conduction of action potentials in the peripheral nerves of frog.