Setup: a A collimated laser beam is delivered into the setup by a beam splitter (BS) and onto a galvanometric scanning mirror (GSM), which is imaged into the back focal plane of an air objective (OBJ1). Scanning the GSM translates the focus in one dimension, as shown by the double-headed arrow in the boxed front focal space of OBJ1. A step mirror reflects the light with different amounts of defocus back into the objective, which then travels through the 4F system onto the GSM, which removes the lateral-scan motion, leaving only defocus in the wavefront. The GSM is again imaged onto the back focal plane of a water dipping objective (OBJ2). As OBJ1 and OBJ2 are pupil matched, OBJ2 forms an aberration-free image of the focus (as formed by OBJ1) in the sample space. b Zoomed-in view of the boxed region from a. The panel on the left shows the focus of the light at its nominal focus. Black arrows show returning marginal rays after reflection. Each step on the mirror results in a focus spot in the sample plane with a displaced axial position. c Alternative configuration with a tilted mirror that allows continuous axial scanning. Here, the remote objective OBJ1 is slightly shifted off the optical axis to create a tilted focus that is perpendicular to the mirror surface. Scanning this focus laterally results in a change in focus, as illustrated by the black arrows

a PSFs obtained using a remote mirror with three steps. b PSFs generated from discrete lateral scanning steps over a planar, tilted mirror. Green arrows depict the zoomed-in view. c Plot showing the full width at half maximum (FWHM) of the PSFs at different axial focus positions. The FWHM of the PSF calculated in the X-direction (red) and Y-direction (black). d Continuous axial scanning obtained by driving the GSM with a triangular waveform at 100 Hz using a tilted, planar mirror. Scale bar: a–d 10 µm. The scale bar in the zoomed-in image of b is 1 µm

a PSFs obtained using a remote mirror with three steps and a sinusoidal scan motion generated by a resonant galvanometer mirror driven at 12 kHz. b Scanning over the same step mirror with a sinusoidal scan motion whose amplitude was adjusted to yield evenly illuminated laser foci. c Sinusoidal scan motion over a planar mirror inclined by 7.5°. d Same arrangement as in c, but with a threefold increased scanning amplitude and a mask to truncate the scan range optically. e Axial intensity profiles of the beams shown in c and d. XZ maximum intensity projections of transmission data are shown in a–d. Scale bar: 10 µm

a Schematic principle of ASLM: a thin light sheet is scanned in its propagation direction, and only the region within the beam waist (red and green bars) is read out by an sCMOS camera. b Point spread function in the XZ plane. c, d RPE cells labelled with GFP-vimentin, imaged with ASLM with a 50 ms integration time. XZ and YZ maximum intensity projections are shown. e, f The same cell imaged with ASLM at 5 ms integration time. XZ and YZ maximum intensity projections are shown. g Genetically encoded multimeric nanoparticles (GEMs) inside two MV3 cells, as imaged by ASLM over a 20 ms image integration time, and 3.57 volumes per second. h, i Axial YZ view of the perinuclear region at two timepoints. Yellow circles indicate detected vesicles, and blue lines illustrate cumulative tracks. Scale bars: b, h, and i 1 µm; d and g 10 µm

a Point spread function using 200 nm fluorescent nanospheres moved to different axial positions, acquired via resonant (12 kHz) remote focusing with a tilted planar mirror (5° tilt). b Rendering of a fixed brain slice labelled with Thy1-GFP acquired using resonant remote focusing. c Axial XZ view of the brain slice shown in b using remote focusing. d Axial XZ view of the same brain slice but acquired with conventional Z-stepping. e Lateral XY view of the brain slice shown in b, acquired with resonant remote focusing. Arrows mark individual spines. The image was convolved with a Gaussian filter (sigma = 1 pixel) to suppress spurious noise. f Schematic drawing of a zebrafish embryo. g Zoomed-in view of the zebrafish heart. The blue plane depicts the axial imaging plane. h Averaged (over 30 cycles) XZ cross-section of the zebrafish heart labelled with Tg(kdrl:EGFP), acquired with a frame rate of 45 Hz. i Kymograph of the beating heart measured along the line shown in h. The kymograph uses raw data, and no averaging was applied. j Volumetric imaging of the zebrafish heart at a volume rate of 156 Hz, XY view with depth encoded in colour. Scale bars: a and e: 5 µm; a (inset): 2 µm; c, h, and j: 20 µm; i: 0.5 s