Easily scalable multi-color DMD-based structured illumination microscopy.

Structured illumination microscopy (SIM) achieves super-resolution imaging using a series of phase-shifted sinusoidal illumination patterns to down-modulate high spatial-frequency information of samples. Digital micromirror devices (DMDs) have been increasingly used to generate SIM illumination patterns due to their high speed and moderate cost. However, a DMD micromirror array's blazed grating structure causes strong angular dispersion for different wavelengths of light, thus severely hampering its application in multicolor imaging. We developed a multi-color DMD-SIM setup that employs a diffraction grating to compensate the DMD's dispersion and demonstrate super-resolution SIM imaging of both fluorescent beads and live cells samples with four color channels. This simple but effective approach can be readily scaled to more color channels, thereby greatly expanding the application of SIM in the study of complex multi-component structures and dynamics in soft matter systems.

Distinct Sub- to Superdiffuse Insulin Granule Transport Behaviors in ß-Cells Are Strongly Affected by Granule Age.

Intracellular transport is a complex process that is difficult to describe by a single general model for motion. Here, we study the transport of insulin containing vesicles, termed granules, in live MIN6 cells. We characterize how the observed heterogeneity is affected by different intracellular factors by constructing a MIN6 cell line by CRISPR-CAS9 that constitutively expresses mCherry fused to insulin and is thus packaged in granules. Confocal microscopy imaging and single particle tracking of the granule transport provide long trajectories of thousands of single granule trajectories for statistical analysis. Mean squared displacement (MSD), angle correlation distribution, and step size distribution analysis allowed identifying five distinct granule transport subpopulations, from nearly immobile and subdiffusive to run-pause and superdiffusive. The subdiffusive subpopulation recapitulates the subordinated random walk we reported earlier (Tabei, 2013; ref 18). We show that the transport characteristics of the five subpopulations have a strong dependence on the age of insulin granules. The five subpopulations also reflect the effect of local microtubule and actin networks on transport in different cellular regions. Our results provide robust metrics to clarify the heterogeneity of granule transport and demonstrate the roles of microtubule versus actin networks with granule age since initial packaging in the Golgi.

Tandem aberration correction optics (TACO) in wide-field structured illumination microscopy.

Structured illumination microscopy (SIM) is a powerful super-resolution imaging technique that uses patterned illumination to down-modulate high spatial-frequency information of samples. However, the presence of spatially-dependent aberrations can severely disrupt the illumination pattern, limiting the quality of SIM imaging. Conventional adaptive optics (AO) techniques that employ wavefront correctors at the pupil plane are not capable of effectively correcting these spatially-dependent aberrations. We introduce the Tandem Aberration Correction Optics (TACO) approach that combines both pupil AO and conjugate AO for aberration correction in SIM. TACO incorporates a deformable mirror (DM) for pupil AO in the detection path to correct for global aberrations, while a spatial light modulator (SLM) is placed at the plane conjugate to the aberration source near the sample plane, termed conjugate AO, to compensate spatially-varying aberrations in the illumination path. Our numerical simulations and experimental results show that the TACO approach can recover the illumination pattern close to an ideal condition, even when severely misshaped by aberrations, resulting in high-quality super-resolution SIM reconstruction. The TACO approach resolves a critical traditional shortcoming of aberration correction for structured illumination. This advance significantly expands the application of SIM imaging in the study of complex, particularly biological, samples and should be effective in other wide-field microscopies.