Optical projection tomography (OPT) is a new tool for three-dimensional (3D) imaging of small tissues or embryos, based on multi-angle recording of internal fluorescent signals using intact whole mount tissue or fish. To understand the route of infection, gills of Y. ruckeri infected rainbow trout were labeled with fluorescent antibody and visualized in 3D by the OPT scanner. Rainbow trout were infected with Y. ruckeri O1 biotype 1 (1 x 109 cells/ml) for 1 hour at 18 °C, and then transferred to clean water. Three fish were sampled at 12 different time points and fixed in 4% PFA. The gills were incubated whole with rabbit anti-Y. ruckeri polyclonal antibody and Alexa Fluor®594 conjugated goat anti-rabbit IgG. After embedding in 1% low melting point agarose, specimens were dehydrated in 100% methanol and cleared in BABB (benzyl alcohol: benzyl benzoate) for OPT scanning. 3D imaging results showed that Y. ruckeri were observed in connection with the primary gill filaments, at the base of gill arches. In contrast, no bacteria were found in control fish gills. More detailed analyses are being processed.