STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution

Abstract

We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 μm deep penetration into biological tissue.

Figure 1

Excitation optics and sample fluorescence of SPIM (A, top) and the STED-SPIM method introduced here (B, bottom). The dots in the cartoon represent individual fluorophores, which are either fluorescent (orange) or nonfluorescent (gray). The green region represents the SPIM excitation sheet and the red regions (in B) represent the dual-sheet STED beam in the sample area.

Figure 2

Comparison of SPIM and STED-SPIM on an ATTO647-stained control fiber sample. The lateral (A) and axial (C) details and contrast of the SPIM image are far less resolved than those achieved with STED-SPIM (B and D). A profile analysis of the same regions revealed consistent 11–40% improvements in lateral images and a 46% improvement in axial images (Fig. S2). Scale bar: 10 μm.

Figure 3

Comparison of SPIM and STED-SPIM on an actin in zebrafish embryo stained with ATTO647 phalloidin. The axial details and contrast of the SPIM image (A) are far less resolved than those achieved with STED-SPIM (B). A profile analysis of the same regions revealed a consistent 30% improvement in axial images (Fig. S3) and 17% improvement in lateral images (Fig. S4). Scale bar: 20 μm.