When making time-lapse movies in multiple fluorescent channels, how to format the data can be very confusing. Each microscope is different in what its software prefers to export.
The bottom line is we can work with pretty much anything except proprietary formats. But we have our preferences.
MetaVi Labs preference order is:
Each channel is in a separate mp4 file (using H.264 compression internally). The images are gray-scale. Keeping the channels separate will provide more accuracy than pre-composited images. MetaVi Labs software will composite and colorize the images for display after the tracking.
Each channel is associated with one folder, and each folder has a collection of tiff files, one file per time point.
Each time point is contained in a multi-frame tiff file. Each file has one image from each of the channels. Each image is grey-scale.