A reproducible imaging protocol should include four main detailed sections. The first should describe the sample preparation and include details about the tissue and/or cell culture preparation, the staining procedure, the optical grade of the coverslip, and the type of mounting media used to mount the sample. The second section should describe the configuration and components of the microscope and include the type of stand, stage, illumination, and detector, as well as the emission (EM) and excitation (EX) filters, objective, and immersion medium specifications. Specialized microscopes may have other important components in the optical path to include. The third section should describe the settings used to acquire an image like the exposure and/or dwell time, final magnification and optical resolution, the pixel and field of view (FOV) sizes, time intervals for any time lapse, total power at the objective (i.e., directed at your sample) and number of planes and step size used to collect a 3-dimensional image, and order of operations used in multi-dimensional image acquisitions. The final section should include details about the image analysis workflow such as the image processing steps, segmentation and measurement methods used to extract information from the image, data size, and necessary computing hardware and networking requirements if data sets are >1 GB, as well as citations and versions for the software and code used to perform any of these steps. Every effort should be made to make an example dataset with accurate metadata available online. Finally, specifics about the type of replicates included in the experiment and details about the statistical analysis conducted are also necessary.
