Fig. 1 |. A schematic of excitation optical paths for both vertical and inclined illumination.

a, Schematic of the microscope setup for both the inclined and the vertical illumination in SPEED microscopy. A 488-nm (blue) and a 568-nm (green) laser are directed into the objective in vertical illumination or offset so that the lasers pass through the focal plane at an angle of θ₃. The inset depicts a diagram of the edge excitation optics. The refractive angle (θ_i) at the interface of two different mediums with different refractive indexes (n_i). The θ₃ depends on the focal length of the objective (a), the refractive indexes of the different mediums in the optical path and the distance (d) between the center and edge excitation beams. The relationship between θ₃ and d follows the equation: $d=a*\tan \left[{\sin }^{-1}\left(\frac{n_{cell}}{n_{oil}}\sin {\theta }_3\right)\right]$, where a = 300 μm, n_cell = 1.33 and n_oil = 1.516. For example, to obtain θ_{3 =} 45°, d needs to be 237 μm. b, An illustration of a molecule traveling in three dimensions as it passes through an NPC. A 2D projection of the same pathway is depicted in the lower panel. c, An illustration of the effect of the optical chopper on laser intensity. The chopper is calibrated to be open for 1/10 of frames captured.