Approaching Maximum Resolution in Structured Illumination Microscopy via Accurate Noise Modeling.

Biological images captured by microscopes are characterized by heterogeneous signal-to-noise ratios (SNRs) due to spatially varying photon emission across the field of view convoluted with camera noise. State-of-the-art unsupervised structured illumination microscopy (SIM) reconstruction algorithms, commonly implemented in the Fourier domain, do not accurately model this noise and suffer from high-frequency artifacts, user-dependent choices of smoothness constraints making assumptions on biological features, and unphysical negative values in the recovered fluorescence intensity map. On the other hand, supervised methods rely on large datasets for training, and often require retraining for new sample structures. Consequently, achieving high contrast near the maximum theoretical resolution in an unsupervised, physically principled, manner remains an open problem. Here, we propose Bayesian-SIM (B-SIM), an unsupervised Bayesian framework to quantitatively reconstruct SIM data, rectifying these shortcomings by accurately incorporating known noise sources in the spatial domain. To accelerate the reconstruction process, we use the finite extent of the point-spread-function to devise a parallelized Monte Carlo strategy involving chunking and restitching of the inferred fluorescence intensity. We benchmark our framework on both simulated and experimental images, and demonstrate improved contrast permitting feature recovery at up to 25% shorter length scales over state-of-the-art methods at both high- and low-SNR. B-SIM enables unsupervised, quantitative, physically accurate reconstruction without the need for labeled training data, democratizing high-quality SIM reconstruction and expands the capabilities of live-cell SIM to lower SNR, potentially revealing biological features in previously inaccessible regimes.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here, we address the role of the cortical microtubules in parasite motility, invasion and egress by utilizing a previously generated mutant (dubbed 'TKO') in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ∼80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization was further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication was not affected. In a 3D Matrigel matrix, the TKO mutant moved directionally over long distances, but along trajectories that were significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory did not impact either movement speed in the matrix or the speed and behavior of the parasite during entry into and egress from the host cell.

The cortical microtubules of Toxoplasma gondii underlie the helicity of parasite movement.

Motility is essential for apicomplexan parasites to infect their hosts. In a three-dimensional (3-D) environment, the apicomplexan parasite Toxoplasma gondii moves along a helical path. The cortical microtubules, which are ultra-stable and spirally arranged, have been considered to be a structure that guides the long-distance movement of the parasite. Here we address the role of the cortical microtubules in parasite motility, invasion, and egress by utilizing a previously generated mutant (dubbed "TKO") in which these microtubules are destabilized in mature parasites. We found that the cortical microtubules in ~ 80% of the non-dividing (i.e. daughter-free) TKO parasites are much shorter than normal. The extent of depolymerization is further exacerbated upon commencement of daughter formation or cold treatment, but parasite replication is not affected. In a 3-D Matrigel matrix, the TKO mutant moves directionally over long distances, but along trajectories significantly more linear (i.e. less helical) than those of wild-type parasites. Interestingly, this change in trajectory does not impact either movement speed in the matrix or the speed and behavior of the parasite's entry into and egress from the host cell.

Decoding the hydrodynamic properties of microscale helical propellers from Brownian fluctuations.

The complex motility of bacteria, ranging from single-swimmer behaviors such as chemotaxis to collective dynamics, including biofilm formation and active matter phenomena, is driven by their microscale propellers. Despite extensive study of swimming flagellated bacteria, the hydrodynamic properties of their helical-shaped propellers have never been directly measured. The primary challenges to directly studying microscale propellers are 1) their small size and fast, correlated motion, 2) the necessity of controlling fluid flow at the microscale, and 3) isolating the influence of a single propeller from a propeller bundle. To solve the outstanding problem of characterizing the hydrodynamic properties of these propellers, we adopt a dual statistical viewpoint that connects to the hydrodynamics through the fluctuation-dissipation theorem (FDT). We regard the propellers as colloidal particles and characterize their Brownian fluctuations, described by 21 diffusion coefficients for translation, rotation, and correlated translation-rotation in a static fluid. To perform this measurement, we applied recent advances in high-resolution oblique plane microscopy to generate high-speed volumetric movies of fluorophore-labeled, freely diffusing Escherichia coli flagella. Analyzing these movies with a bespoke helical single-particle tracking algorithm, we extracted trajectories, calculated the full set of diffusion coefficients, and inferred the average propulsion matrix using a generalized Einstein relation. Our results provide a direct measurement of a microhelix's propulsion matrix and validate proposals that the flagella are highly inefficient propellers, with a maximum propulsion efficiency of less than 3%. Our approach opens broad avenues for studying the motility of particles in complex environments where direct hydrodynamic approaches are not feasible.

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

Multicolor structured illumination microscopy and quantitative control of polychromatic light with a digital micromirror device.

Linear structured illumination microscopy (SIM) is a super-resolution microscopy technique that does not impose photophysics requirements on fluorescent samples. Multicolor SIM implementations typically rely on liquid crystal on silicon (LCoS) spatial light modulators (SLM's) for patterning the excitation light, but digital micromirror devices (DMD's) are a promising alternative, owing to their lower cost and higher speed. However, existing coherent DMD SIM implementations use only a single wavelength of light, limited by the lack of efficient approaches for solving the blazed grating effect for polychromatic light. We develop the requisite quantitative tools, including a closed form solution of the blaze and diffraction conditions, forward models of DMD diffraction and pattern projection, and a model of DMD aberrations. Based on these advances, we constructed a three-color DMD microscope, quantified the effect of aberrations from the DMD, developed a high-resolution optical transfer function measurement technique, and demonstrated SIM on fixed and live cells. This opens the door to applying DMD's in polychromatic applications previously restricted to LCoS SLM's.

Bad metallic transport in a cold atom Fermi-Hubbard system.

Strong interactions in many-body quantum systems complicate the interpretation of charge transport in such materials. To shed light on this problem, we study transport in a clean quantum system: ultracold lithium-6 in a two-dimensional optical lattice, a testing ground for strong interaction physics in the Fermi-Hubbard model. We determine the diffusion constant by measuring the relaxation of an imposed density modulation and modeling its decay hydrodynamically. The diffusion constant is converted to a resistivity by using the Nernst-Einstein relation. That resistivity exhibits a linear temperature dependence and shows no evidence of saturation, two characteristic signatures of a bad metal. The techniques we developed in this study may be applied to measurements of other transport quantities, including the optical conductivity and thermopower.

Spin-imbalance in a 2D Fermi-Hubbard system.

The interplay of strong interactions and magnetic fields gives rise to unusual forms of superconductivity and magnetism in quantum many-body systems. Here, we present an experimental study of the two-dimensional Fermi-Hubbard model-a paradigm for strongly correlated fermions on a lattice-in the presence of a Zeeman field and varying doping. Using site-resolved measurements, we revealed anisotropic antiferromagnetic correlations, a precursor to long-range canted order. We observed nonmonotonic behavior of the local polarization with doping for strong interactions, which we attribute to the evolution from an antiferromagnetic insulator to a metallic phase. Our results pave the way to experimentally mapping the low-temperature phase diagram of the Fermi-Hubbard model as a function of both doping and spin polarization, for which many open questions remain.

Phase Separation and Pair Condensation in a Spin-Imbalanced 2D Fermi Gas.

We study a two-component quasi-two-dimensional Fermi gas with imbalanced spin populations. We probe the gas at different interaction strengths and polarizations by measuring the density of each spin component in the trap and the pair momentum distribution after time of flight. For a wide range of experimental parameters, we observe in-trap phase separation characterized by the appearance of a spin-balanced core surrounded by a polarized gas. Our momentum space measurements indicate pair condensation in the imbalanced gas even for large polarizations where phase separation vanishes, pointing to the presence of a polarized pair condensate. Our observation of zero momentum pair condensates in 2D spin-imbalanced gases opens the way to explorations of more exotic superfluid phases that occupy a large part of the phase diagram in lower dimensions.