It is known that 3-D imaging enhances the ability to perform delicate endoscopic maneuvers, such as dissection or precise suturing. Moreover, it provides observers with a better understanding of spatial anatomy. We propose here a 3-D polarization-type endoscopic imaging system, which has three features of stereo endoscopic image processing features: real time image capture, retrieval, and presentation of left and right images on a TFf-LCD monitor. We also compare the established 2-D type, the electric shutter-type 3-D imaging, and the new polarizationtype 3-D endoscopic imaging systems in terms of the performance of laparoscopic tasks in an animate situation. The polarization-type 3-D system is shown to be superior to the electric shutter-type in image quality, stability of performance, and speed based on the testing of suturing and loop passing.

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Fluo-3, a fluorescent Ca²⁺ indicator with lower Ca²⁺ affinity and absorbance of longer wave length than fura-2, the most popular indicator, was applied to fresh brain slice preparations. Although fluo-3 was confirmed to be a good indicator for intracellular Ca²⁺ concentration in cultured hippocampal cells and also in organotypic culture of brain slices, it had unfortunate properties in the fresh slice preparations. When hippocampal slice preparation stained with fluo-3 in the acetoxymethyl form (fluo-3/AM) was stimulated by a depolarizing medium (50 mM KCI), the fluorescence of the slice preparation did not increase, but decreased, which indicated a decrease in Ca²⁺ concentration. The fluorescence also decreased markedly when the preparation was exposed to Ca²⁺-free medium (containing 1 mM EGTA), which had no effect on the fluorescence intensity of the slice preparation stained with fura-2/AM. The present results suggested that in the fresh slice preparation de-esterified or partially de-esterified form of fluo-3 might remain in the extracellular space conjugating to the dead cells or to the surface of the living cell membrane as a result of its hydrophobic properties, and thus detect the changes in extracellular Ca²⁺ concentration. This is an important warning against the use of such dyes to stain fresh slice preparations.

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We cultured mycelia of the edible mushroom Pleurotus ostreatus (Fr.) Kummer Y-1 in YMG medium containing 15 mM Cs and 10,000 Bq/kg ¹³⁷Cs and evaluated the proliferation of mycelia and their ¹³⁷Cs accumulation. The dry weight of mycelia increased until 72 h after initiation of culture but was relatively constant thereafter until 120 h of culture (2.7-3.1 as the weight ratio to 48 h after initiation of culture). These results suggested that P. ostreatus Y-1 mycelia are in the logarithmic phase until 48 h after initiation of culture but are in the stationary phase after 72 h or more. The ¹³⁷Cs accumulation expressed as the concentration ratio (¹³⁷Cs concentration in the dried mycelia/¹³⁷Cs concentration in the wet medium) was already about 10 after 24 h of culture, indicating early uptake. Elementary analysis of P. ostreatus Y-1 mycelia in the proliferation and stationary phases was performed using a scanning electron microscope (SEM)-energy dispersive X-ray microanalyzer (EDX). The morphology of the mycelia was similar between the two phases; the mycelia tip protruded in a gentle curve. The ratio of Cs in the stationary phase to that in the proliferation phase (stationary/ proliferation phase Cs ratio) at the mycelial root dense with mycelia formed in the early stage was about 5 times that at the mycelial tip. The stationary/logarithmic phase ratio of K or P, essential elements of mushrooms, did not markedly differ between the mycelial tip and root, but the amount of each element was slightly larger in the proliferation phase than in the stationary phase.

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We devised a closed chamber apparatus in order to examine growth cones in cultured neurons for several hours at high magnification. The chamber apparatus consisted of a stainless steel frame, a piece of bottom cover glass on which neurons were cultured, and a piece of roof cover glass with two φ 0.3 mm holes. The holes were sealed with adhesive tape after the chamber was filled with culture medium. The chamber apparatus was prepared on an inverted differential-interference-contrast microscope. The elongation of neurites and the movement of filopodia in growth cones were recorded with a hypersensitive video camera, an image processor, and a time-lapse video recorder.

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We describe the newest version of the protocol to construct a bacterial artificial chromosome (BAC) library which carries uniform and larger insert DNA. This protocol is based on the five major improvements reported by Osoegawa et al. (1998) to increase the cloning efficiency and decrease the level of non-insert clones. The first improvement is a purification of high molecular weight (HMW) DNA with a pulse field gel electrophoresis, which eliminates inhibitory contaminants for restriction enzymes and leads to the increase of digestion efficiency of HMW DNA. The second is the doublesize fractionation method, in which partially digested DNA is subjected to an electrophoresis in reverse direction to remove smaller DNA fragments, and then size-fractionated in forward direction. This step enables the preparation of uniform and large-size insert DNA without contamination by smaller DNA. The third is recovering the size-fractionated DNA from agarose gel by the use of electroelution, rather than the use of β agarase as in conventional methods. This step dramatically increases both the purity and integrity of large DNA fragments, leading to an increase of cloning efficiency. The fourth step is the concentration of the ligation products using dialysis against polyethylene glycol buffer with high osmotic pressure. This step increases the number of the transformants produced in a single transformation trial, resulting in saving time and cost. The fifth is the most effective improvement in preparing a BAC vector to minimize a level of non-insert clones. This step employs the ligase-treatment of vector fraction to completely remove the molecules with phosphorylated ends.
In this paper, we introduce the precise optimal conditions in each step, which enabled us to make BAC libraries with around 200 kb insert DNA. One example is the RPCI23 library constructed from C57BL/6J female mice. This library consists of approximately 180,000 clones with an average insert size of 197 kb and contains only 6.8 % noninsert clones. This library covers the mouse genome 11.2 times with the most uniform and largest insert DNA except for YAC libraries.

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We investigated the intracellular dynamics of osteopontin (OPN) in living osteoblastic cultured cells by the dual-channel confocal microscopy of OPN-GFP fusion proteins and R6 (rhodamine B hexyl ester)-stained organelles in osteoblastic UMR-106 cells. Here we present the detailed experimental procedures.

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