Figure 1: Depiction and assembly of the chamber.

(a) 3D drawing of the different parts of the chamber, comprising the main chamber frame (1) sized accordingly to a microscopy slide, the bottom coverslip (2) that will allow imaging from inverted microscopes, the agarose pad (3) that is imprinted according to the chosen type of stamp corresponding to the sample of interest (round or elongated for example), the PDMS seal (4) that ensures the ceiling of the chamber and prevents any contact between the content of the chamber and the printed resin and finally the frame of the lid (5) in which the coverslip is slid to allow imaging from top for upright microscopes configuration. (b) Assembly of the main chamber frame (1) with the coverslip and the PDMS seal. The most important step here is to clip properly the PDMS in the chamber to ensure no further leakage of the agarose or the medium. (c) After the adjunction of 2 mL of warm LMP (Low Melting Point) agarose the stamp is disposed to prevent the formation of bubbles. (d) After a cooling step (better is to keep the device at 4 °C for a few minutes), the stamp is removed carefully. Ideally the agarose should be maintained with a thick and smooth device such as for example insect forceps, and if necessary, few drops of medium can be disposed to facilitate the detachment of the agarose from the stamp. (e) After removal of the stamp the chamber can be filled with the appropriate medium. If necessary at this step, or with the lid on top (f) of the assembly procedure, the device can be placed in the incubator to allow the medium to diffuse in the agarose and to equilibrate in gas. After having loaded the samples within the holes, the chamber can be placed on the stage of the microscope (g).