Figure 6.

Simultaneous multicolor excitation-multiplexed microscopy of up to six different cellular structures in HeLa cells using four excitation lasers at 470, 514, 561, and 640 nm. (a–g) Spectrally unmixed data from a five-color sample are shown, with Golgi complex (a), lipid droplets (b), lysosomes (c), mitochondria (d), and nucleus (e), with composite overlay (f) and volumetric (g) images. (h–o) Spectrally unmixed data from a six-color sample, adding actin (h) to the existing components, are shown: Golgi complex (i), lipid droplets (j), lysosomes (k), mitochondria (l), and nucleus (m), with composite overlay (n) and volumetric (o) images. The magenta and red arrows in (j), (k), and (n) identify distinct lipid droplets and lysosomes, respectively. Scale bars, 10 μm. All images were acquired with 1-ms pixel dwell times, covering 512 × 512 pixels. To see this figure in color, go online.