Analytical Sciences

Simultaneous Imaging of Multiple Fluorescent Probes in Bio-Cells

Hideaki MATSUOKA,* Yuri KOSAI,* Mikako SAITO,* Norihide TAKEYAMA,** and Hiroshi SUTO***

*Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
**Genesia Corporation, 4-12-1-301, Shimo-Renjaku, Mitaka, Tokyo 181-0013, Japan
***National Astronomical Observatory of Japan, 650 North A'ohoku Place, Hilo, Hawaii, USA

In order to obtain the full spectrum from 400 to 800 nm of each pixcel of a microscopic image, a unique spectro-imaging system was developed using an image slicer. The image slicer is composed of 100 photo fibers which are arranged in a matrix of 10 x 10 at the entrance and 100 x 1 at the exit. A line of this 100 signals is passed through a glism and projected onto a CCD. This system was applied to the fluorescent imaging of bio-cells. One of the demonstrative examples was simultaneous measurements of the Ca²⁺ concentration and the pH using of respective fluorescent probes. An electric signal was applied to BY-2 protoplasts and the fluorescent spectrum from 500 nm to 800 nm was measured every 5 s. The spectrum of the BY-2 protoplasts changed in response to the electric signal and the Ca²⁺ concentration, and the pH changes could be monitored. The wavelength resolution was satisfactory, but the space resolution was still rough in comparison with the usual microscopic systems. Notwithstanding these conditions, we could obtain discrete data from more than several tens of sites in a single-cell or a chain of several cells.

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