Fig. 1

a Drosophila (left), larval zebrafish (centre) and adult mouse schematics (right) with central nervous system highlighted (green) to illustrate size differences. Insets next to the mouse for size-comparison on the same scale. b Optical configurations of standard diffraction limited (DL, left) 2P setup with parallel laser beam entering objective’s back aperture. Right, non-telecentric (nTC, middle, right) configurations use a diverging laser beam instead. As a result, the field of view and focal distance are expanded, and the point spread function (PSF) elongates. These effects scale with the angle of divergence (compare nTC₁ and nTC₂). c Typical neuronal somata in species shown in (a), as interrogated by 2P setups shown in (b), respectively. d In vivo 7 dpf larval zebrafish (HuC:GCaMP6f) imaged with a Sutter-MOM DL setup at full field of view (top) and when zoomed in to reveal individual neuronal somata (bottom) as indicated. e same zebrafish as (d), next to two further zebrafish imaged using nTC₂ configuration at maximal field of view (top). Zooming in to the same area as in (d, bottom) reveals cellular detail (e, bottom). f In vivo adult mouse cranial window over somatosensory cortex imaged with nTC₂ maximal field of view (top) and when zoomed in as indicated (bottom). g Left: Optical configuration of a standard DL setup with collimation system consisting of a scan lens and a tube lens to set-up an infinity collimated laser beam at the objective’s back aperture. Effective refractive power and relative distances of lenses indicated. The intermediary focal point (IFP) is behind the scan lens (arrowhead). Middle: nTC₁ configuration replaces the scan lens with two plano-convex lenses (L1,2). The relative position of L2 to the tube lens defines the position of the IFP, which is now further along the laser path. As a result, the field of view can be expanded to between 1.2 and 1.8 mm. Right: nTC₂ configuration using a single plano-convex lens (L3) allows FOV expansion to 2.5–3.5 mm. h complete nTC setup, including an ETL positioned in front of the scan mirrors for rapid axial-scanning. PMTs, Photomultipliers. i FOV expansion under nTC combines two effects: Increased focal distance (left) and reduced numerical aperture (N.A., right), which together yield a larger effective focal plane and enlarged PSF. j Power at sample measured for all configurations, expressed as a percentage of the power that reaches the scanning mirrors. k point spread functions (PSFs) measured for all configurations, with size of typical neuronal somata of different species indicated. All scale-bars 10 µm. l, m, lateral (l) and axial (m) spread of the PSFs quantified. Errors in mean ± s.d., n = 20 experimentally independent samples per measurement. Data leading to j, l, m in Source data file.