Fig. 12.

A fluorescence photoactivation pulse-escape experiment. Images excerpted from a time-lapse movie of a cultured DRG neuron in a long-term myelinating co-culture. The neuron is co-expressing mCherry (to permit observation of the axons prior to photoactivation) and PAGFP-NFM. The culture was stained with FluoroMyelin^™ Red, which is a vital fluorescent marker for compact myelin (Monsma and Brown, 2012). A, B. The mCherry fluorescence is shown in blue to distinguish it from the FluoroMyelin^™ Red fluorescence, which is shown in red. The myelin sheath appears purple in A because of FluoroMyelin^™ Red cross-talk on the mCherry channel. We avoid mCherry cross talk on the FluoroMyelin^™ Red channel by using a custom filter that excites the FluoroMyelin^™ Red with blue light, taking advantage of this dye’s unusually large Stokes shift. C. The PAGFP-NFM fluorescence (green) and FluoroMyelin^™ Red fluorescence (red) before photoactivation. D, E. Images of the PAGFP-NFM fluorescence at t = 0 and 120 min after photoactivation. F. Graph of the average pulse-escape kinetics for 15 axons. Note that a significant fraction of the fluorescence remains in the activated region after two hours, reflecting the prolonged pausing behavior of these polymers. Scale bar = 15 μm. Data from Monsma et al. (2014).