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. 2019 Jun;16(6):501-504.
doi: 10.1038/s41592-019-0401-3. Epub 2019 May 6.

Epi-illumination SPIM for Volumetric Imaging With High Spatial-Temporal Resolution

Free PMC article

Epi-illumination SPIM for Volumetric Imaging With High Spatial-Temporal Resolution

Bin Yang et al. Nat Methods. .
Free PMC article


We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Conflict of interest statement

Conflict of interests

A patent application has been filed covering the reported microscope design.


Figure 1.
Figure 1.
Optical setup and spatial resolution of the system. (A) Schematic of the experimental setup. O1 is used for both illumination (shown in green) and fluorescence (shown in yellow) detection. A remote imaging module composed of two objective lenses O2 and O3 is used to image the oblique plane. A water container (shown in dark blue) in the focal space of O2 and O3 separates them by a glass coverslip, with one side being air medium and the other side water. Left inset shows the definition of the coordinate system. Right inset shows the detailed design of the remote imaging module. (B-C) PSF of the eSPIM with Gaussian and Bessel excitations, measured with 45 nm green fluorescent beads. Representative cross-sections of the PSF in the x’y-plane, in the yz’- plane and intensity plots along the three axes of a bead are shown. The FWHMs are 362.4 nm, 285.1 nm and 533.8 nm along the x’-, y- and z’- axes with Gaussian excitation, and are 346.3 nm, 309.5 nm, 408.9 nm along the three axes with Bessel excitation. (D-E) eSPIM images with Gaussian and Bessel excitations of a mixture of HEK 293T cells with endogenous clathrin A or lamin A/C labeled by mNG211 knock-in. Maximum intensity projection of the 3D dataset in the xy-plane is shown together with representative cross-sections in the xz-plane and in the yz-plane. B and C are raw data without deconvolution. D and E are data after deconvolution using the Richardson-Lucy algorithm with 10 iterations. At least five experiments were repeated independently with similar results for B-E.
Figure 2.
Figure 2.
Parallel volumetric live imaging of cells in multiwell plates. (A) HEK 293T cells were endogenously labeled with split mNeonGreen2. 3D views of a representative time point for each well are shown at the top. Four zoomed-in 3D views are shown at the bottom. The box sizes are 93.1 μm × 93.1 μm × 16.0 μm for the sixteen full field of view images, and 26.6 μm × 26.6 μm × 16.0 μm for the four zoomed-in images. Three experiments were repeated independently with similar results. (B) Drosophila S2 cells stably expressing mRFP-actin were treated with DMSO or Cytochalasin D, an inhibitor of actin polymerization. Representative max intensity projection images of the cells with different treatments and at different time points are shown. Right-top panel shows the normalized surface areas of the cells as a function of time. Right-bottom graph shows the surface areas change of the cells after treatment relative to before treatment. Values are averaged from three experiments, with error bars indicating the standard deviation. Green dots show all the data points.

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