Simultaneous Dual-Color Imaging of mRNA and Ribosomes

MEFs were imaged on a previously described custom-built microscope (Grünwald and Singer, 2010 (link); Katz et al., 2012 (link)). Simultaneous dual-color imaging experiments of mRNA and ribosomes were performed with an Olympus (Waltham, MA) 1.45 NA 150X objective with a resulting pixel size of 107 nm. Cobolt (San Jose, CA) Jive 50TM 561 nm and Cobolt Calypso 100 491-nm lasers were coupled into a single optical fiber, collimated, and delivered through the back port of the Olympus IX-71 stand. A tunable lens was inserted into the light path to produce objective base total internal reflection fluorescent excitation. A 405 nm laser (Coherent, Santa Clara, CA) was free-space-coupled into the back port of the microscope stand for full field photo-activation. Activation was manually controlled with a UNIBLITZ shutter (Vincent Associates, Rochester, NY) and purposely kept low (~1.5% of total possible output) so only a few ribosomes were fluorescently activated for single particle tracking. Imaging experiments were run through MetaMorph Software (Molecular Devices, Sunnyvale, CA).