Whole brain imaging of larval zebrafish with XLFM.
(a) Schematic of XLFM. Lenslet array position was conjugated to the rear pupil plane of the imaging objective. Excitation laser (blue) provided uniform illumination across the sample. (b–c) Point sources at two different depths formed, through two different groups of micro-lenses, sharp images on the imaging sensor, with positional information reconstructed from these distinct patterns. (d) Maximum intensity projections (MIPs) on time and space of time series volume images of an agarose-restrained larval zebrafish with pan-neuronal nucleus-localized GCaMP6f (huc:h2b-gcamp6f) fluorescence labeling. (e) Normalized neuronal activities of selected neurons exhibited increasing calcium responses after the onset of light stimulation at t = 0. Neurons were ordered by the onset time when the measured fluorescence signals reached 20% of their maximum. (f) Selected neurons in (e) were color coded based on their response onset time. Scale bar is 100 μm.

Example of camera captured raw imaging data of larval zebrafish.
Raw fluorescence imaging data consisted of 27 sub-images of a larval zebrafish formed by 27 micro-lenses. Under the condition that the PSF is spatially invariant, which is satisfied apart from small aberrations, the algorithm can handle overlapping fish images.

Dependence of imaging resolution on the sparseness of the sample.
Characterization of the dependence of imaging resolution on the sparseness of the sample using computer simulation. (a) Maximum intensity projections (MIPs) of a numerically simulated (top) and reconstructed (bottom) larval zebrafish with randomly distributed active neurons. Red and green lines indicate positions where simulated (red) and reconstructed (green) cross-sections are compared. We assumed that the total number of neurons in the zebrafish brain is 80,000, and gradually increased the sparseness index ρ, the fraction of neurons activated at a given frame. (b–d) Characterization of the reconstruction results for different ρ. Insets are magnified views of rectangular regions. Red and green dots are simulated and reconstructed neurons, respectively.

Characterization of the autofocus system.
(a) Autofocus camera behind a one-dimensional lenslet array captured triplet images of the fish head (up). Its autocorrelation function was computed (bottom). (b) Central line profile of the autocorrelation function was extracted and inter-fish distance was computed as local maximums in the autocorrelation function. (c) Axial shift of the fish head, calibrated by moving the piezo at a constant interval, changed linearly (red line) with inter-fish distance.

3D tracking of larval zebrafish.
(a) Representative time varying error signals in three dimensions, defined as the difference between real head position and set point. Inset provides magnified view at short time interval. Lateral movement can be rapidly compensated for within a few milliseconds with an instantaneous velocity of up to 10 mm/s. The axial shift was small compared with the depth coverage (200 μm) during whole brain imaging, and thereby had minor effect on brain activity reconstruction. (b) Tracking images at four time points during prey capture behavior, acquired at low (left) and high (right) magnification simultaneously. Scale bars are 1 mm (left) and 200 μm (right). (c) Kinematics of behavioral features during prey capture. Shaded region marks the beginning and end of the prey capture process.

Whole brain imaging of larval zebrafish during prey capture behavior.
(a) Renderings of whole brain calcium activity at six time points (up) and the corresponding behavioral images (bottom). Features used to quantify behavior were: fish-paramecium azimuth α; convergence angle between eyes β; head orientation γ; and fish-paramecium distance d. (b) Maximum intensity projections of zebrafish brain with pan-neuronal cytoplasm-labeled GCaMP6s (huc:gcamp6s). Boundaries of four brain regions are color marked. (c) Neural dynamics inferred from GCaMP6 fluorescence changes in these four regions during the entire prey capture behavior (up) and the kinematics of behavioral features (bottom). Note that between t2 and t4, fish-paramecium distance d exhibits three abrupt kinks, representing the three attempts to catch prey.