Figure 4.

Single particle tracking in 3D of a GSH-Qrod using polarized optics. (A) Fluorescence images of a single GSH-Qrod at various Z-positions ranging from −800 to 800 nm. Each image is the sum of the P- and S-polarization images at one Z-position. (B) FWHM values of the images in A as a function of the Z-position along the X axis (red line) and Y axis (blue line). (C) Ellipticity as a function of the Z-position at various anisotropies (−0.60 to 0.59). (D) Calculated precision in the position of Qrods along the X, Y, and Z axis (red, blue, and green symbols) as a function of the Z-position. Error bars represent standard deviations. (E) Test tracking with 20 nm steps when the Z-position of the Qrod was near zero. A GSH-Qrod fixed on a coverslip was moved (using two Piezo actuators) at discrete 20 nm steps once every 1 s simultaneously along the X (red line), Y (blue line), and Z axis (green line). The frame rate of the CCD camera was 10 frames/s. Standard deviations of the tracking for 10 s (excluding the stepping moments) were 5, 7, and 19 nm along the X, Y, and Z axis, respectively.