Figure 2.

Stability Characterization and Noise-Level Estimation

(A) Characteristic interference pattern of a fluorescent microsphere displaced relative to the focal plane of the detection objective.

(B) Radial intensity profile averaged over all angles as a function of the imposed objective height. This profile serves as a look-up table (LUT) to quantify z movements of the microsphere during microscope rotation. The dashed line indicates the fit position for the profile shown in (A).

(C) Performance test of the z tracking algorithm. A control signal drives a piezo to displace the detection objective in steps of 0.5 μm (red), and the relative distance of the microsphere to the objective (blue; error bars indicate SD; N = 5) is computed by fitting the instantaneous intensity profile of the microsphere onto the LUT shown in (B).

(D) Characterization of the shift along the z direction of the scanned volume as a function of the microscope rotation angle (blue; error bars indicate SD, N = 5). Rotation direction is from −20° to +20° in blue and the reverse rotation is in red. The inset shows the corresponding movements in the x-y plane. The schematic defines the coordinate system.

(E) Step stimulation protocol used for estimating the mechanically induced noise level.

(F) Distribution of the intensity change per pixel induced by a −15° microscope rotation relative to the intensity value measured just before the step (RFP- [red] and GCaMP6s- [blue] expressing fish). Light colors correspond to the intrinsic noise distribution measured in the absence of microscope rotation. Data were centered and normalized via Gaussian fits.

(G) Mean mechanically induced noise level (Δσ) measured with RFP- (red) and GCaMP6s- (blue) expressing fish as a function of microscope angle (error bars indicate SD; N = 3 fish).