Publications & technical resources

Stress granules are mRNA-protein granules that form when translation initiation is limited, and they are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures, referred to as cores, within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions and links between stress granules and human diseases and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently, with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly, and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Super-resolution localization microscopy involves acquiring thousands of image frames of sparse collections of single molecules in the sample. The long acquisition time makes the imaging setup prone to drift, affecting accuracy and precision. Localization accuracy is generally improved by a posteriori drift correction. However, localization precision lost due to sample drifting out of focus cannot be recovered as the signal is originally detected at a lower peak signal. Here, we demonstrate a method of stabilizing a super-resolution localization microscope in three dimensions for extended periods of time with nanometer precision. Hence, no localization correction after the experiment is required to obtain super-resolved reconstructions. The method incorporates a closed-loop with a feedback signal generated from camera images and actuation on a 3D nanopositioning stage holding the sample.

Laser scanning microscopes are helpful tools for the visualization of 3D structures at the submicron scale. By pointwise raster scanning the sample with a tightly focused laser spot, they collect sample information in a sequential manner. We present a scanning microscope that has the ability to record 3D sample information from a single 2D scan. By combining camera detection with double-helix phase engineering in the emission path, the microscope allows for postacquisition refocusing within an axial range of roughly 400 nm with a high-NA lens (NA 1.35). Refocused images are extracted from a single 2D data set by applying different synthetic pinholes, i.e., by integrating over a small predefined area in the image acquired for every sampling point. 3D cross-sectional images of a stained microtubule network within fixed African green monkey kidney cells are presented, demonstrating the capability of the system. The imaging paradigm enables faster data acquisition with potentially lower phototoxic side effects.

Single-particle tracking has been applied to study chromatin motion in live cells, revealing a wealth of dynamical behavior of the genomic material once believed to be relatively static throughout most of the cell cycle. Here we used the dual-color three-dimensional (3D) double-helix point spread function microscope to study the correlations of movement between two fluorescently labeled gene loci on either the same or different budding yeast chromosomes. We performed fast (10 Hz) 3D tracking of the two copies of the GAL locus in diploid cells in both activating and repressive conditions. As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes. We found that under repressive conditions, the GAL loci exhibited significantly higher velocity cross-correlations than they did under activating conditions. This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation. We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

Precise imaging of the cell surface of fluorescently labeled bacteria requires super-resolution methods because the size-scale of these cells is on the order of the diffraction limit. In this work, we present a photocontrollable small-molecule rhodamine spirolactam emitter suitable for non-toxic and specific labeling of the outer surface of cells for three-dimensional (3D) super-resolution (SR) imaging. Conventional rhodamine spirolactams photoswitch to the emitting form with UV light; however, these wavelengths can damage cells. We extended photoswitching to visible wavelengths >400 nm by iterative synthesis and spectroscopic characterization to optimize the substitution on the spirolactam. Further, an N-hydroxysuccinimide-functionalized derivative enabled covalent labeling of amines on the surface of live Caulobacter crescentus cells. Resulting 3D SR reconstructions of the labeled cell surface reveal uniform and specific sampling with thousands of localizations per cell and excellent localization precision in x, y, and z. The distribution of cell stalk lengths (a sub-diffraction-sized cellular structure) was quantified for a mixed population of cells. Pulse-chase experiments identified sites of cell surface growth. Covalent labeling with the optimized rhodamine spirolactam label provides a general strategy to study the surfaces of living cells with high specificity and resolution down to 10–20 nm.

When a single molecule is detected in a wide-field microscope, the image approximates the point spread function of the system. However, as the distribution of molecules becomes denser and their images begin to overlap, existing solutions to determine the number of molecules present and their precise three-dimensional locations can tolerate little to no overlap. We propose a localization scheme that can identify several overlapping molecule images while maintaining high localization precision. A solution to this problem involving matched optical and digital techniques, as here proposed, can substantially increase the allowable labeling density and accelerate the data collection time of single-molecule localization microscopy by more than one order of magnitude.

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

This article surveys the recent application of optical Fourier processing to the long-established but still expanding field of single-molecule imaging and microscopy. A variety of single-molecule studies can benefit from the additional image information that can be obtained by modulating the Fourier, or pupil, plane of a widefield microscope. After briefly reviewing several current applications, we present a comprehensive and computationally efficient theoretical model for simulating single-molecule fluorescence as it propagates through an imaging system. Furthermore, we describe how phase/amplitude-modulating optics inserted in the imaging pathway may be modeled, especially at the Fourier plane. Finally, we discuss selected recent applications of Fourier processing methods to measure the orientation, depth, and rotational mobility of single fluorescent molecules.

Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule super-resolution imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule super-resolution imaging. We highlight recent methods based on quadrated pupil imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.