Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination (original) (raw)

We demonstrate a simple and efficient method for producing ultrathin Bessel ('non-diffracting') light sheets of any color using a line-shaped beam and an annulus filter. With this robust and cost-effective technology, we obtained two-color, 3D images of biological samples with lateral/axial resolution of 250 nm/400 nm, and high-speed, 4D volume imaging of 20 μm sized live sample at 1 Hz temporal resolution. The twin goals of high spatial and high temporal resolutions in live imaging of biological samples have been elusive until the recent progress in light-sheet microscopy 1–6 , especially the groundbreaking invention of the lattice light-sheet microscope (LLM) 7 in which 'non-diffracting' ultrathin light sheets are employed. The unparalleled capability to perform live imaging of objects ranging in size from molecules to embryos with high spatiotemporal resolution and minimal phototoxicity, has attracted much attention. The crafting of an ultrathin lattice light-sheet (LL) starts from writing a 2D optical lattice pattern onto a spatial light modulator (SLM). Incident light diffracted by this SLM is then filtered by an annular ring mask (" annulus ") to give the 'non-diffracting' Bessel property. The optical lattice pattern is then dithered to form a desired lattice light sheet. For each excitation light frequency, a different pattern is written onto the SLM, and for multicolor imaging, the lattice pattern must be rapidly switched and synchronized to the color changes. Despite the many advantages of this method, the efficiency in producing the LL is very low: less than 1% of the light gets to the sample, and the source of this inefficiency can be traced to the fast-switching SLM. Here we demonstrate a technique, Line Bessel Sheet (LBS) (Fig. 1), to produce ultrathin 'non-diffracting' Bessel sheets without the cost, complexity and inefficiency of the SLM. The excitation light is made line-shaped (e.g. by passing through a slit, or other means, see Supplementary Note 1) and then passes through an annulus imaged to the back focus plane of the excitation objective (Special Optics, numerical aperture (N.A.) 0.7). The line image on the sample will be in focus for an extended length 8,9 along the propagation direction, forming a 'non-diffracting' Bessel sheet (Fig. 1a, Supplementary Note 1) 10–12. We demonstrate that it is possible to design a combination of line thickness and annulus size so that input laser light of any color will emerge as ultrathin light sheet of that color (Supplementary Movie 1) without dithering and without switching any components or mask patterns. As the excitation ultrathin light sheet optical setup is totally passive, no complex synchronization is required other than the change of laser color with the camera frame for sequential multicolor imaging. Simultaneous multicolor imaging is also feasible. With the exception of the illumination arm, the main body of our microscope is constructed following the design of the LLM 7 with some modifications (Supplementary Figure 1a). We designed two line thickness-annulus combinations: LBS1 and LBS2, each with different characteristics (Supplementary Note 1). In LBS1 (Fig. 1b), the central light sheet thickness is matched to the axial resolution of the detection objective (600 nm), maximizing the detection efficiency and minimizing photo-bleaching. This ultrathin light sheet maintains its shape for over 15 μ m, compared to 3 μ m for a Gaussian light sheet of the same 600 nm thickness (Fig. 1f). In LBS2 (Fig. 1c), we crafted a thinner central sheet (~400 nm), which results in higher intensity side lobes. With proper deconvolution,