Figure 1.

Single molecule imaging using PALM technique. (A) Diagram of the TIRF microscopy setup. By angling the excitation light beam though a combination of the refractive indices of the lense, oil, coverslip, and aqueous media, the beam is reflected at the glass/media interface and is directed back into the objective. This creates an evanescent wave at the interface which effectively excites fluorophores only about 100 nm into the sample. Therefore only those fluorescent proteins or dyes within that region will be excited and emit fluorescence which can be detected. (B) Demonstrating the difference between EGFP and PA-GFP. EGFP will absorb and emit light without prior activation before bleaching off. PA-GFP needs to be activated by short-wavelength light before it is confirmationally able to absorb excitation and emit light. (C) (left) Localized and rendered PALM image of BK channels on the plasma membrane of a HEK293 cell (scale bar: 5 μm) with a region of interest (right) showing the localization of molecules in detail (scale bar: 1 μm).