(93a) Improvement and Calibration of a System for Ma-Tiraf Microscopy

Understanding of the interaction between cells and surfaces is important for cellular adhesion, signaling, and the formation of biofilms. Multiple angle total internal reflection aqueous fluorescence (MA-TIRAF) microscopy can be a useful tool for measuring distances (~10-100nm) between cells and surfaces while maintaining the cells' free motility. The resulting information can then be applied to analyzing the cell-surface interaction.

TIRAF microscopy has been previously used to observe bacterial cells adhering to and swimming along both uncoated glass and treated glass surfaces. This information was used to elucidate the forces acting to maintain swimming bacteria along the glass surface. The technique of MA-TIRAF marries TIRAF microscopy, first described by Gingell et al, with the ability to alter the illumination depth of the view field. The addition of multiple-angle capabilities allows the penetration depth of the evanescent wave over the course of a measurement to be varied; this provides an additional form of distance measurement. Using the two techniques together should allow more accurate results over a greater range of distances than previously possible.

The current work is focused on the construction and calibration of an improved system for MA-TIRAF microscopy. An optic fiber was precisely aimed and used to direct the laser beam at the sample. This fiber is positioned at different angles using a novel motorized yoke. Fine adjustment screws were installed in the supports of this yoke to provide the high precision z-direction adjustment required to achieve a stationary focal point for the laser as it moves through different angles. A laser angle calibrator was created to allow determination of the exact angle of laser incidence and to calibrate the computer control for the aiming system. A sample chamber was designed which is capable of containing either flowing or stationary bacterial solution. Part of this sample chamber was also designed to align and maintain the position of the semi-cylindrical prism used to transmit the laser at multiple angles. It is anticipated that the system will be tested for its ability to generate valid data for a single angle soon, followed rapidly by expansion to its full multiple angle capability.