Figure 4

3D reconstructions from different samples prior and after deconvolution. The deconvolution approach described in this paper provides a significant increase in sharpness and in the level of details of light sheet microscopy recordings for different samples and magnifications ranging from 1x to 20x. (a) 3D reconstructions (MIP projection) of an E12.5 mouse embryo that was immune-stained and chemically cleared according to³². Nerve fibers are highlighted by NF-160 fluorescence labelling³². The reconstruction was obtained from 667 slices recorded using a 2.5x objective (Zeiss FLUAR 2.5x, Carl Zeiss, Germany) with an NA of 0.12 and a 0.5x post magnification. Recording was performed using a light sheet microscope equipped with a single cylindrical lens of 80 mm focal length and a 6 mm wide slit aperture as described in³. For imaging, a Cool Snap Cf CCD camera (Roper Scientific, Germany) with 1392 × 1040 pixel resolution was used. Illumination time: 430 ms. (a1) The left column shows a 3D-reconstruction obtained from the unprocessed raw data. The right side shows a reconstruction obtained from the same data set after deconvolution with a calculated PSF. (a2) Zoomed details from three selected regions before deconvolution (a1–c1) and after deconvolution (a2–c2). Deconvolution parameters were NA = 0.14, λ_ex = 488 nm, λ_em = 520 nm, n = 1.561, f = 80 mm, d = 8 mm, stop criterion = 0.1%, no damping. (b) 3D reconstructions obtained from an isolated EGFP expressing mouse hippocampus that has been chemically cleared according to³³. (b1) Reconstruction obtained from 1050 slices recorded using a 5x objective (FLUAR 5x, Carl Zeiss, Germany) with a NA of 0.25 and a 0.5x post magnification (left image) and the same data set after deconvolution (right image). Recording was performed with a light sheet microscope equipped with a 80 mm cylindrical lens and a 6 mm wide slit aperture as described in³. For deconvolution, the data set was split into 1 × 1 × 3 equally sized blocks. Deconvolution parameters were NA = 0.25, λ_ex = 488 nm, λ_em = 520 nm, n = 1.561, f = 80 mm, d = 8 mm, stop criterion = 0.1%, no damping. Imaging was done using a Cool Snap cf camera (Roper Scientific, Germany) with 1392 × 1040 pixel resolution. Illumination time: 2000 ms. (b2) Reconstruction obtained from 221 slices of 1392 × 1040 pixel resolution recorded using a 20x objective (LUCPLFLN 20x, NA 0.45, Olympus, Germany) (left column) and the same data set after deconvolution (right column). Recording was performed with a light sheet microscope equipped with a 80 mm cylindrical lens and a 16 mm wide slit aperture as described in³. Deconvolution parameters were NA = 0.45, λ_ex = 488 nm, λ_em = 520 nm, n = 1.561, f = 80 mm, d = 16 mm, 30 iterations. Imaging was done using a Cool Snap cf (Roper Scientific, Germany) camera with 1392 × 1040 pixel resolution. Illumination time: 10000 ms. c) Part of the head of an entire adult mouse chemically cleared according to³⁴. c1) Reconstruction obtained from 1297 slices recorded using a 2x objective (XLFLUOR 2x, Olympus, Germany), with an NA of 0.14 and a 0.63x post magnification (left image) and the same data set after deconvolution (right image). Recording was performed using a light sheet microscope equipped with a modified light sheet generator as described in²². For imaging an Andor Neo CMOS camera (Andor, Ireland) with 2560 × 2160 pixel resolution was used. Illumination time: 180 ms. Deconvolution parameters were NA = 0.14, λ_ex = 488 nm, λ_em = 520 nm, n = 1.561, f = 80 mm, d = 16 mm, 30 iterations, no damping. c2) Zoomed details from regions a-b marked in c1 before deconvolution (left) and after deconvolution (right). d) Cortical neurons recorded in an entire mouse brain that was chemically cleared according to³⁵. (d1) Reconstructions obtained from 777 slices recorded using a 4x objective (XLFLUOR 4x, Olympus, Germany) with an NA of 0.28 and a 2x post magnification (left column) and the same data set after deconvolution (right column). Recording was performed using a light sheet microscope with a modified light sheet generator as described in²². Imaging was done using an Andor Neo CMOS camera with 2560 × 2160 pixel resolution. Illumination time: 250 ms. Deconvolution parameters were NA = 0.28, λ_ex = 488 nm, λ_em = 520 nm, n = 1.561, f = 80 mm, d = 16 mm, 30 iterations, stop criterion = 0%, no damping. (d2) Two different zooms of d1 before deconvolution (left) and after deconvolution (right).