Figure 1 ∣. Microscope design.

(a) A laser amplifier beam (1030 nm; ~280 fs pulse duration) passes through a chopper synchronized to the triangle-wave laser-scanning pattern (Supplementary Figs. 1,2) and is given uniform intensity across a microlens array (lenslet spacing: 100 μm), which creates N_x × N_y beamlets of equal intensity. N_x·N_y is the number of lines scanned in the specimen. Compound (L1 and L4) and aspherical lenses (L2, L3 and L5) establish intermediate and specimen focal planes (red dots: beamlet foci) as Fourier-conjugates to the plane of the scanning mirror. Fluorescence returns through the objective lens, reflects off a dichroic mirror, and is focused by a tube lens (L6) and a relay lens pair onto a sCMOS camera synchronized to the scanning mirror. Excitation and emission pathways for 3 example foci are shown in red and green, respectively. Inset: Bottom, The beam-shaper comprises an expander, a flattop shaper, and a reducer. Top, Beam intensity profiles at each stage.

(b) To scan images at rates ≤1 kHz, the N_x × N_y beamlet array is projected onto the specimen at an angle, θ = tan⁻¹(1/N_x), between the (X_S, Y_S) camera-frame coordinate system (dashed rectangle) and the (X_M, Y_M) microlens array coordinate system. We generally used ≤20 × 20 laser foci (15 μm spacing between beams) oriented 2.9° to the scanning direction, yielding 0.75 μm between scanned lines. We cropped the illumination to match the area projected onto the camera.