抄録
To study Ca signals in situ in realtime, we developed a fiber-coupled confocal microscope, and observed fluorescently-labeled cells in the brain of anesthetized rat, or cultured cells. The microscope system consisted of DPSS laser (488 nm, 200 mW), microlens-attached Nipkow disk scanner (CSU10, Yokogawa Co), and a lens-coupled imaging fiber unit. The imaging unit had over 20,000 of single mode fiber of 3 micron in diameter, which was scannned with Nipkow disk to form a confocally sectioned image. The effective NA value was approximately 0.2. The thickness of the sectioned image was < 5 micron as estimated from the HWHM of the fluorescence intensity measured with the focus shift (over a fluorescent bead of 10 micron in diameter). The fiber unit was 2.2 mm thick, for easier approach to the target inside the brain without a large physical damage. The confocal images were captured with an electron multiplying-CCD camera at over 30 F/s. For intravital imaging, we inserted the fiber unit into the brain, in which some neurons were labeled with Calcium orange. When the incidental face of the fiber unit was placed 3 mm below the brain surface, many bright spots and fibrous structures were clearly discernible. They showed fluctuation in fluorescence intensity partly dependent on the breathing and possibly on some neuronal activities. Such an in situ imaging technique is very promising for detailed studies on the relationship between neuronal structure and functions. [J Physiol Sci. 2006;56 Suppl:S246]
