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Even Illumination Multi-Angle Total Internal Reflection Fluorescence Microscopy

Thursday, November 07, 2013 — Poster Session II

12:00 p.m. – 2:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • Y Fu
  • G. H. Patterson


An even illumination multi-angle total internal reflection fluorescence (EI-MA-TIRF) microscope was constructed to achieve super-resolution in axial direction. Two-axles galvo mirrors were added to a TIRF system and controlled by a waveform generator to generate a hollow cone incident beam. The total internal refection in continuous azimuthal directions resulted in a uniform evanescent field for illumination that produced TIRF images free of interference fringes and shadow artifacts. The incident beam angle was adjusted by manipulating the amplitudes of two sinewave outputs from the waveform generator and controlled the penetration depth of the evanescent field. Selective z-sectioning TIRF was achieved by decreasing incident beam angles towards the critical angle. Layer-by-layer TIRF images from the sample above the surface of the coverslip were obtained by multi-angle selective photobleaching each layer’s fluorphores sequentially. The 3D TIRF image was reconstructed by stacking these layer-by-layer TIRF images together. Using this method, 3D TIRF images of actin-filaments with around 20 nm apart each layer was obtained. Two-color such 3D TIRF images were used to differentiate two protein structures close to each other within 20 nm in axial direction.

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