Supplementary Figure 11: Comparison of photobleaching observed with a spinning-disk confocal microscope and with the eSPIM system. | Nature Methods

Supplementary Figure 11: Comparison of photobleaching observed with a spinning-disk confocal microscope and with the eSPIM system.

From: Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution

Supplementary Figure 11

For both microscopes, volumetric fluorescence images of HEK 293T cells with endogenous clathrin light chain A labeled by mNeoGreen211 knock-in were recorded. The exposure conditions were adjusted so that similar signal levels were obtained on both microscopes. When setting up the parameters of the spinning-disk confocal microscope so that the SNR is comparable to that of the eSPIM, we focused on the bottom of the cell where the features are the sharpest. The pixel intensity of all slices (36 slices for spinning disk and 51 for eSPIM, adjacent slices separated by 400 nm in both cases) within each volumetric dataset is summed, normalized to the first volume and then plotted as a function of the volume number. The data are fitted with a double exponential decay function. Inset shows the plot of the first 150 data points. The first volumetric images in both modes have approximately the same signal-to-noise ratio. The objective used in the confocal experiment is Nikon CFI Plan Apo VC 100× Oil (NA = 1.40). The pixel sizes are, respectively, 127.6 nm and 133.0 nm for the confocal and SPIM images. Three experiments were repeated independently, with similar results.

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