Additional file 1:

The Selective Plane Illumination Microscopy (SPIM) setup. A: Schematics of the SPIM system. Lateral (B) and top (C) views of the SPIM setup. The green path corresponds to the illumination axis and the perpendicular red path corresponds to the detection axis. The sample is suspended through the theta-stage (θS) in the physiological chamber (PC) and is positioned in the focal plane of the detection objective. It can be moved in the x, y and z axis and rotated (θ axis, blue arrow in c). T: telescope; M: mirror; CL: cylindrical lens; TL: tube lens; IO: illumination objective; PC: physiological chamber; XS: X-stage; YS: Y-stage; ZS: Z-stage, θS: θstage; F and FW: filter wheel. D: The physiological chamber was designed using the Open source Blender software (top) and then manufactured by stereolithography of a photosensitive epoxy resin (bottom). The two tinny holes on the top of the chamber (red circles) allow to inject CO2 directly in the physiological chamber at the surface of the culture medium.

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Additional file 2:

SPIM images of a spheroid of Capan-2 human pancreatic cancer cells labelled with DRAQ5™. A: Raw images corresponding to the XY optical sections at the indicated depths inside the spheroid. Scale bar 50 μm. The white arrows show dividing cells located at several cell layers of depth inside the spheroid. B: The XY plane at 130 μm depth is shown with the XZ and ZY planes, parallel to the detection axis, at the Y and X positions indicated by the dashed lines (Scale bar 50 μm). The inserts correspond to the enlargement of the region in the white square on the XY section that displays a mitotic cell. Scale bar 5 μm. The progressive loss of signal observed along the x-axis results from light scattering and absorption by the spheroid that attenuates the light sheet illumination. Horizontal stripes parallel to the light sheet (x-axis) are sample-dependent artifacts specific to SPIM technology. C: 3D visualisation of a multiview reconstruction of four stacks recorded at various angles (0-315°) at incremental steps of 90°. The corresponding stack is shown in Additional file 14.

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Additional file 3:

Three-dimensional SPIM imaging of a Capan-2 cell spheroid labelled with DRAQ5™. This movie shows a z-stack of 250 slices at a slice spacing of 1 μm. Arrows show mitotic cells. Laser 595 nm; illumination objective 10× NA = 0.25; detection objective 10× NA = 0.3. Scale bar 50 μm.

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Additional file 4:

Three-dimensional SPIM imaging of an H2B-HcRed-expressing spheroid. Movie showing two merged z-stacks at 0° and 180°. For each z-stack, 200 slices were recorded with a slice spacing of 1 μm. Arrows show mitotic cells.

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Additional file 5:

Three-dimensional SPIM imaging of mitoic cell inside an H2B-HcRed-expressing spheroid. This movie shows 28 slices through a mitotic cell. The images correspond to a region from the z-stack shown in Additional file 4.

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Additional file 6:

Three-dimensional visualization of two regions of the stack shown in Additional file5. Arrows show mitotic cells. This visualization was obtained with the 3D viewer plug-in of Fiji software.

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Additional file 7:

3D reconstruction of a mitotic cell inside an H2B-HcRed-expressing spheroid. Visualization of the 3D reconstruction of the stack shown in the right part of the Additional file 6 (blue isosurfaces, interphase nuclei; red isosurfaces, mitotic condensed chromosomes).

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Additional file 8:

Sample holder preparation for time-lapse acquisitions. A: Sample holders were prepared using a 1.25 ml Combitip from which the tip has been removed. A Phytagel solution (10 g/l in PBS) is aspirated in the Combitip and formed after polymerisation the sample holder. B: The sample holder (shaded grey) was uncast by applying gentle pressure then suspended on a plunger for transfer into the physiological chamber of the microscope. The plunger was made from a Combitip with the tip cut off to leave an empty space (light grey). C: Enlargement of the Phytagel sample holder showing the cavity generated by the shape of the tip of the Combitip plunger. Culture medium was placed in this cavity, in which a spheroid can grow. For more details on sample holder preparation an illustrated protocol is available for downloading here: http://www.ip3d.fr/IP3D/SPIM/SPIM.html webcite

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Additional file 9:

SPIM imaging of a live spheroid. SPIM imaging of a live spheroid expressing H2B-HcRed. Z-stacks of 100 slices at slice spacing of 1 μm were recorded every three minutes (10× objective, NA = 0.3). The maximum projection of the z-stacks is shown for each time point.

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Additional file 10:

3D visualization of the division of two cells. Three-dimensional visualization of an enlarged region of Additional file 9 showing the progress of two cells through mitosis. This visualization was obtained with the 3D viewer plug-in of Fiji software. Arrows show the two dividing cells.

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Additional file 11:

3D reconstruction of the division of two cells. Visualisation of the isosurfaces corresponding to the 3D reconstruction of the mitotic chromosomes (red) and interphase nuclei (blue) in a region of the stack shown in Additional file 9.

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Additional file 12:

SPIM imaging of a live H2B-HcRed-expressing spheroid treated with Paclitaxel. Z-stacks of 200 slices at slice spacing of 1 μm were recorded every three minutes. For each time point, the maximum projection of an enlarged region of each stack is shown (10× objective, NA = 0.3). Arrows indicate mitotic cells.

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Additional file 13:

3D reconstruction of arrested mitotic cells following taxol treatment. Visualisation of the isosurfaces corresponding to the 3D reconstruction of a region of the stack shown in Additional file 12.

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Additional file 14:

Stack of the registered and fused multiviews through a spheroid stained with DRAQ5. Single planes of the stack of the reconstructed spheroid showed in the Additional file 2.

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