Extended Data Fig. 1: Schematic of Oblique Line Scanning microscope for single-molecule tracking.

(a) Schematic of the OLS microscope, based on scanning an inclined excitation light sheet using galvanometric scanning mirrors across a sample placed in an inverted microscope. The OLS microscope is based on a multiwavelength optical excitation provided by a Laser Engine Module and coupled to the beam shaper by a collimator-coupled single-mode fiber. The beam shaper transforms the incoming Gaussian-shaped optical excitation to an optical light-sheet that is focused into the back focal plane of the microscope’s objective lens along the light sheet’s line axis and scanned along the scan axis using galvanometric mirrors. The resulting oblique light sheet is sent into a water-immersion-coupled and environmentally-controlled sample holding plate, whereas the relative position of the microscope’s focal plane is controlled by an autofocus unit. The excited fluorescence is spectrally filtered from the excitation light by dichroic filters and emission filters and projected onto a high-speed sCMOS camera. Synchronization of optical excitation, scanning, and acquisition is achieved by a custom-build control unit. (b) The autofocus unit is based on detecting a 780 nm-LED reflection on the top surface of the sample-holding glass bottom and repositioning the objective lens to ensure appropriate focal plane positioning within the sample. (c) The optical confocal scanning mode is achieved by scanning an inclined and focused light sheet through the objective’s focal plane. Background suppression is achieved by confocal arrangement of the inclined light sheet (green), the objective’s depth of field, and synchronized rolling shutter detection (orange). (d) The beam shaping subassembly projected along the line axis (x) and the scan axis (y) shapes collimated optical excitation into a light sheet by a series consisting of a Powell lens, cylindrical lenses, a spherical lens, and a planoconvex lens before encountering the (e) The optical line scanning framework is based on an inclined light sheet in the sample plane achieved by focusing the optical excitation along the line axis in the objective’s back focal plane and positioning the optical excitation along the scan axis at an offset position relative to the optical axis of the objective. The corresponding detection of optically-aligned fluorescence is projected onto the camera sensor. (f) The OLS acquisition mode relies on detecting fluorescence by matching the camera’s area of exposed pixels and synchronizing the camera’s rolling shutter to the optically-projected intensity line of fluorescence excited by the inclined light sheet. Abbreviations: A, Aperture; AF, Autofocus; AFS, Autofocus sensor; Cam, Camera; CL, Cylindrical lens; Col, Colimator; DM, Dichroic mirror; DoF, Depth of field; EF, Emission filter; ExP, Exposed Pixels; FB, Fly-Back; Fluo, Fluorescence; GB, Glassbottom; Inc, Incubator; LED, Light emitting diode; LEM, Laser engine module; M, Mirror; MIC, Microscopy illumination control; OL, Objective lens; OLS, Oblique line scan; PC, Personal computer; PL, Planoconvex lens; POWL, Powell lens; RU, Run-up; SL, Spherical lens; SM, Scanning mirror; SMF, Single-mode fiber; SS, Sample stage; ST, Scantime; TaC, Trigger and control; WI, Water immersion.