High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

Abstract

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work we present a multi-plane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multi-plane image splitting prism combined with high-speed SR-SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally super-resolved three-dimensional image stack. We demonstrate the performance of multi-plane SR-SIM by applying this instrument to the dynamics of live mitochondrial network.

Mapping volumes to planes: Camera-based strategies for snapshot volumetric microscopy

“…The depicted 4-plane prism consists of two isosceles trapezoidal prisms joined along their base; the connecting surface is coated to serve as a 50: 50 BS, and total internal reflection at the interface of the glass prism with the surrounding air replaces the mirrors. The majority of design considerations take place before manufacturing, making it an easy-to-implement and mechanically stable configuration with almost no chromatic aberration over the visible spectral range, enabling diffractionlimited imaging [3,10,33,34]. The assembly of a compact image splitter is also possible by gluing together different off-the-shelf components, such as BS cubes and right angle prisms [11], see Figure 3D.…”

“…The first implementations focused on fluorescence single-particle tracking of beads and small organelles, as well as single receptor proteins [22,35,36]. Later, different super-resolution microscopy modalities were realized (localization microscopy [29], (live-cell) superresolution optical fluctuation imaging [10,22,37], and livecell structured illumination imaging [33]). The systems can be adapted for single-cell and whole organism imaging, e.g.…”

Simulating digital micromirror devices for patterning coherent excitation light in structured illumination microscopy

“…They also offer high switching speeds, they can handle high light intensities, and depending on coating, are not sensitive to light polarization. This makes them an interesting option for many SLM applications in microscopy [14][15][16][17][18][19][20][21][22]. However, the jagged nature of the micromirror array gives rise to the blazed grating effect that becomes rather annoying and detrimental when using DMDs in combination with a coherent light source [23,24].…”