I was wondering in what ways do you deposit fiducials and what do you use as fiducials. We have tried fluorescent beads (tetra speck) and colloidal gold particles. But I am not really satisfied by both of them, while the tetra specks are bright and nice to use for multi color experiments they are a bit big and most of the times to bright. The gold beads are most often to dim in our hands and therefor don’t always turn up in all channels.
But mostly I am not satisfied with the amount of fiducials I can get on my sample in view. There are either to many, to few or in a place I don’t want them, obscuring the structures of interest. Another problem is to get the fiducials at the focus depth of choise, getting them on the coverslip is rather trivial but a bit above the problems start. Did anybody find solutions to these problems?