Ultraview vox spinning disk microscope

Live-cell microscopic experiments were carried out with a Perkin­Elmer UltraVIEW VoX spinning-disk microscope equipped with a Hamamatsu C9100-23B EMCCD camera and a CFI Apochromat TIRF 100× objective (NA = 1.49). Imaging was performed using agarose-pad slides at room temperature, as described previously (Tran et al., 2004 (link)). For maximum projection imaging, stack images containing 11 planes with 0.5-µm spacing were acquired; for high-temporal-resolution imaging, single-plane images were acquired. MetaMorph v. 7.7 software (www.moleculardevices.com), together with ImageJ v. 1.5 (imagej.nih.gov), was used to analyze microscopic data, to create the kymograph graphs, and to perform the fluorescence intensity measurements. Plot graphs were generated with KaleidaGraph v. 4.5 (www.synergy.com), and statistical analysis was performed with either Microsoft Excel or KaleidaGraph.

Neurons (25–30 DIV) were imaged at room temperature in modified Tyrode solution (pH 7.4) on a Perkin-Elmer UltraView VoX Spinning Disk microscope with an autofocus system, a motorized stage, 488 nm/50 mW and 561 nm/50 mW diode lasers and a Hamamatsu C9100-50 camera. This system included a PhotoKinesis feature for fluorescence recovery after photobleaching that was used in the case of photoswitching experiments. All experiments were performed using a 60X/1.4 numerical aperture (NA) oil immersion objective lens (CFI Plan Apo VC, Nikon), and images were acquired at 4 frames/s. One dendritic and one axonal arborization were randomly selected to perform each experiment.