Sub-micron aperture plate for intracellular calcium transient measurement

We have fabricated a sub-micron aperture plate for localized measurement of intracellular calcium transient at multipoints simultaneously. The sub-micron aperture perforated on the plate surface decreases the excitation area of fluorescence. The aperture array was fabricated by using EB lithography and a lift-off method. The excitation area size was estimated to be less than 500 nm by using fluorescent beads. As a sample specimen, nerve cells from a silkworm moth were cultured directly on the plate. After fluorescent dye loading, cultured nerve cells were excited by the laser light that passed through the sub-micron aperture. [Ca²⁺] influx was induced by adding acetylcholine and glutamate stimulation solution. A consequent rise of intracellular [Ca²⁺] was successfully measured at multi apertures. The fluorescence intensity fluctuation ΔF/F reached 30%. This result showed the validity of our proposed device.