Extended Data Figure 7: LLS spectral imaging and linear unmixing.

a, Schematic of the hardware used for six-colour light sheet microscopy. The specimen was excited using six lasers sequentially, by LLS illumination. Emitted light passed through a series of interference filters and was collected using a sCMOS camera. b, Plot of the emission intensity of the indicated fluorophores as a function of excitation wavelength, in images of singly labelled cells acquired as described in a. To identify fluorophores in the image data, we applied an excitation-side unmixing algorithm (see Image Acquisition and Unmixing). Our multispectral time-lapse LLS images consisted of upwards of 4.9 billion sets of six-colour-channel pixels (547 × 640 pixels per plane × 140 planes per cell × 100 time points per cell × 10 cells). Because the solution to the unmixing operation at every pixel is independent of every other pixel, we distributed the unmixing operation over 32 cores of a computer workstation. c, Plots of mean pixel intensity values for all six fluorophores in every pixel in singly labelled cells that were segmented as foreground. Cells were singly labelled with CFP–SKL, Mito–EGFP, ss-YFP–KDEL, mApple–SiT, Texas Red dextran, or BODIPY 665/676. The error in LLS unmixing is higher than for confocal (see Extended Data Fig. 1f) as expected and is due partly to the fact that only six channels of spectral information were used to unmix the overlapping spectra. n = 149 pixels (CFP), n = 3,910 pixels (EGFP), n = 9,180 pixels (YFP), n = 1,549 pixels (mApple), n = 806 pixels (Texas Red Dextran), n = 3,248 pixels (BODIPY 667/676). Error bars represent s.e.m. d, Tilted volume rendering of the same cell shown in Fig. 3a. Scale bar, 10 μm. e, Zoomed, segmented images from the cell shown in d. The left panel does not include the ER channel while the right panel does (transparent yellow). Scale bar, 5 μm. Micrographs in d and e are representative of 10 cells captured.