Daisuke Oyamatsu

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring.

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate–probe distance

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.

Electrochemical/photochemical formation of enzyme patterns on glass substrates using a scanning electrochemical/confocal microscope

A hybrid system consisting of a scanning electrochemical microscope (SECM) and scanning confocal microscope (SCFM) was fabricated and used for micropatterning and imaging of diaphorase immobilized on a glass substrate. Simultaneous imaging of the diaphorase spots demonstrated that the SECM can provide information on a localized enzyme reaction, while the SCFM affords information on the location of the active enzyme. By using this SECM/SCFM system, spatially selective deactivation of diaphorase was performed by inducing a local electrochemical reaction or by irradiating with the focused laser. The resulting patterns of diaphorase were simultaneously imaged with the SECM/SCFM system.

Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection.

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.