Fig. 5
Volumetric time-lapse imaging with two high-NA views enables investigation of subcellular dynamics at high spatiotemporal resolution. a Single planes at indicated depth from coverslip, showing dynamics of microtubules in Jurkat T cells expressing 3XEGFP-EMTB. Red arrow in upper series highlights inward and subsequent outward displacement of microtubule bundles at base of cell, yellow arrow in lower series shows contemporaneous deformation of the left nuclear edge. b Maximum intensity projections showing response of GFP-labeled mitochondria in U2OS cells to NaN3, an inhibitor of cytochrome c oxidase. c Higher-magnification views of pink dashed rectangular regions shown in b. NaN3 was added at 13 min (shown as orange line between 12.5 and 17.5 min time points). Orange and pink arrows highlight response to NaN3 addition, resulting in inhibition of ATP synthesis through oxidative phosphorylation and triggering fragmentation and circularization of mitochondria. d Representative maximum intensity projections of a three-fold nematode embryo expressing GCaMP3 from the nmr-1 promoter, highlighting calcium transient during backwards movement. Red circle: AVA cell body, green dashed line ventral nerve cord (VNC). e–g Indicate higher-magnification views of VNC, corresponding to green dashed rectangular regions in d. Red and yellow arrows indicate relative positions (posterior, anterior) along VNC. A kymograph of dF/F along the VNC is shown in h, diagonal white arrow emphasizes intensity gradient progressing along space and time. Intensity profiles corresponding to red rectangular region at top are further plotted in i for reference, intensity derived from the AVA cell body is also shown. dF/F derived from the two views and their average are plotted. Gray box denotes period of backwards locomotion. j Higher-magnification view of yellow dashed rectangular region in d. Contrast has been adjusted (saturating cell bodies) in order to emphasize fine spatial details within the embryonic nerve ring. All images are deconvolved, derived from direct and reflected 1.1 NA views. All perspectives are shown relative to the coverslip, except in d, where rotation angle relative to Y axis is shown. All scale bars: 5 μm, except in c, 1 μm. See also Supplementary Movies 9–12
