Figure 4

Me-MPM improved signal-to-noise during time-lapse imaging of photosensitive tissue samples. Images of 250-μm acute brain slices prepared on embryonic day 17 from a mouse embryo that underwent consecutive two-color in utero electroporation of hrGFP and DsRedII to generate two distinct groups of red or green fluorescing cells in the cortex, schematically represented in a. Therefore, any yellow signal should correspond to signal bleed-through (verified empirically using CLSM on independent samples). Tissue slices were imaged using simultaneous ME-MPM at 920 nm and 1015 nm (b) or SE-MPM at 970 nm (c). Note that bleed-through of green signal in the red emission channel was higher for SE-MPM (white arrowheads). Two green and two red cells for each experiment were traced at 1-hour intervals over the 12-hour imaging window (white and grey arrows, respectively) and their movement is shown (right panel in b and c). Note that cell movement was robust for green and red neurons that were imaged using ME-MPM. In similar samples, CLSM has previously been shown to cause more photo-induced cell death [2].