X cite 120

35-mm glass-bottom culture dishes (WillCo 70670-52) containing cells infected with VSV-P-eGFP were placed on the stage of a Nikon Ti inverted microscope fitted with a 60×/NA1.4 oil objective. The microscope was placed on an optical breadboard (Newport) mounted onto 4 pneumatic vibration isolators (Thorlabs PWA075). The stage environment was set to 37 C, 5% CO₂. The light source for fluorescence microscopy was a metal halide short-arc lamp (Lumen Dynamics X-CITE 120). The epi-illumination cube for GFP consisted of a 470/40 nm excitation filter, 495 nm dichroic filter, and 525/50 emission filter (Chroma 49002). Images were digitized to 16 bits at 100 frames per second by a CMOS camera with 6.5 μm x 6.5 μm pixels (PCO Edge 5.5).
Setting the intensity of the excitation light for fluorescence imaging requires a balance between two competing factors. The first is bleaching, which can be reduced by decreasing the source intensity or the duration of image stacks. The second is signal-to-noise ratio for the RNP particle spots, which arises from photon noise and background fluorescence. Fortunately, each RNP particle contains approximately 400 eGFP groups. This allows the competing needs to be met if the duration of the image stacks is limited to about 4 s when the frame rate is 100 fps.

Single-cell static, time-lapse images and movies were captured on an Olympus IX83 inverted microscope using a 100×, 1.3-numerical-aperture phase-contrast objective. Phase-contrast and fluorescence images were obtained with a Hamamatsu ORCA-R2 digital charge-coupled-device camera, and the light source was the Xcite 120 light-emitting diode (Lumen Dynamics, Mississauga, ON, Canada). Emission filters were purchased from Chroma Technology (Bellows Falls, VT). The specific emission filters were DAPI-5060C-OMF (excitation [EX] filter, 377/50 nm; emission [EM] filter, 447/60 nm; dichroic mirror [DM], 409 nm), GFP- 3035D-OMF (EX filter, 473/31 nm; EM filter, 520/35 nm; DM, 495 nm), mCherry-B-OFF (EX filter, 562/40 nm; EM filter, 641/75 nm; DM, 593 nm). Images were processed with the Olympus software package cellSense Dimensions (v 1.14). Data from three biological replicates were analyzed for each strain. Images presented are from a single representative experiment.

Light stimulation was performed on a setup consisting of a Zeiss upright DIC microscope (Examiner A1, Zeiss, Germany). Near UV, blue and green light were generated from the mercury lamp (X-Cite 120, Lumen Dynamics, USA), filtered by fluorescence cubes (UV: 375/28 nm; Green: 546/12 nm; Blue: 470/20 nm, Zeiss Inc., Germany) and provided through the ×40 water-immersion objective. A circular illumination spot around neurons was obtained by passing light through a pin hole and focusing the light with a water immersion ×40 objective.