Single molecule measurements Sample Clauses

Single molecule measurements. ‌ The experimental setup for single-molecule imaging has been described in detail pre- viously [81]. The samples were mounted on an inverted microscope (Axiovert100, Zeiss) equipped with a 100× objective (NA=1.4, Zeiss). The region-of-interest on an ultrasensitive CCD camera coupled to the microscope was set to 50 × 50 pixels. The apparent pixelsize was 220 nm. Measurements were performed by illumination of the samples for 5 ms at 514 nm (Argon-ion laser, Spectra Physics) at an intensity of 2 kW/cm2. The cells were photobleached for a period of 2-5 sec and sequences of 200-500 images with a timelag of 50 ms were taken. Use of an appropriate filter combination (Chroma) permitted the detection of the fluorescence signal on a liquid nitrogen-cooled CCD-camera (Princeton Instruments). The setup allowed us to im- age individual fluorophores at a signal-to-background-noise ratio of ∼30 leading to a positional accuracy of σ0 = 40 nm. The measurements always focused on the apical cell membrane and never lasted longer than 15 - 60 sec per cell and 2 hr in total.
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