Figure 2.

Imaging FCS calibration and sensitivity of soSPIM-FCS. (A) Typical single pixel autocorrelation function for 100 nm tetra-spec fluorescent beads (red) and organic Atto565 dye (gray) in 1 × PBS buffer. Data were acquired with a sCMOS camera using a 32 × 32 (32 × 16 organic dyes) pixel ROI at 155 μs camera integration time. (Dashed lines) Fits of Eq. S1; see the Supporting Material. (B) Dependence of diffusion coefficient, D, at different camera pixel binning and lateral PSF (analysis was performed with varying binning values from 1 × 1 to 5 × 5 pixels). (C) The diffusion coefficient cumulative distribution function for 100 nm fluorescent beads and Atto 565 dyes in 1 × PBS. (D) Dependence of the diffusion time on the effective area (postacquisition camera binning). Inverse slope estimates the diffusion coefficient (D₂₂°C = 3.3 ± 1.3 μm²/s for 100 nm fluorescent beads). Experiments were performed on a sCMOS camera with a pixel size of 160 nm. ACF analysis were performed with varying binning values from 1 × 1 to 5 × 5 pixels. To see this figure in color, go online.