Fig. 4. Total internal reflection fluorescence (TIRF) microscopy of targeting events. (A) Fixed B16F1 cells were stained with monoclonal anti-tubulin antibodies followed by Alexa Fluor-488-labeled secondary antibodies and TRITC-phalloidin. The representative wide-field and TIRF microscopy images show three targeted filopodia (arrowheads), one nontargeted filopodium (asterisk) and microtubules. The nontargeted filopodium, one targeted filopodium and all the microtubules are clearly visible by TIRF microscopy. Two targeted filopodia are not visible by TIRF. (B) Percentage of microtubules (MT) and filopodia (FL) visible by TIRF microscopy during targeting events (targeted) and nontargeting (nontargeted). Fewer targeted filopodia are visible by TIRF compared with nontargeted filopodia (^*P<0.01, unpaired t-test). (C) Comparison of wide-field and TIRF microscopy of targeting events in live cells co-transfected with YFP-fascin and YFP--tubulin. As shown in the wide-field time series, a microtubule (MT1) targets a filopodium (FL3) at 00:00 seconds and another microtubule (MT2) targets a filopodium (FL2) at 00:20 seconds. As shown in the TIRF time series, FL3 and FL2 disappear after targeting. By contrast, the nontargeted filopodium (FL1) remains visible by TIRF throughout the sequence. Bars, 2 µm. Errors bars indicate standard error.