Uniform intensity in multifocal microscopy using a spatial light modulator

Multifocal microscopy (MFM) offers high-speed three-dimensional imaging through the simultaneous image capture from multiple focal planes. Conventional MFM systems use a fabricated grating in the emission path for a single emission wavelength band and one set of focal plane separations. While a Spatial Light Modulator (SLM) can add more flexibility, the relatively small number of pixels in the SLM chip, cross-talk between the pixels, and aberrations in the imaging system can produce non-uniform intensity in the different axially separated image planes. We present an in situ iterative SLM calibration algorithm that overcomes these optical- and hardware-related limitations to deliver near-uniform intensity across all focal planes. Using immobilized gold nanoparticles under darkfield illumination, we demonstrate superior intensity evenness compared to current methods. We also demonstrate applicability across emission wavelengths, axial plane separations, imaging modalities, SLM settings, and different SLM manufacturers. Therefore, our microscope design and algorithms provide an alternative to fabricated gratings in MFM, as they are relatively simple and could find broad applications in the wider research community.Comment: 15 page

Localization precision in chromatic multifocal imaging

Multifocal microscopy affords fast acquisition of microscopic 3D images. This is made possible using a multifocal grating optic, however this induces chromatic dispersion effects into the point spread function impacting image quality and single-molecule localization precision. To minimize this effect, researchers use narrow-band emission filters. However, the choice of optimal emission filter bandwidth in such systems is, thus far, unclear. This work presents a theoretical framework to investigate how the localization precision of a point emitter is affected by the emission filter bandwidth. We calculate the Cram\'er-Rao lower bound for the 3D position of a single emitter imaged using a chromatic multifocal microscope. Results show that the localization precision improves with broader emission filter bandwidth due to increased photon throughput, despite a larger chromatic dispersion. This study provides a framework for optimally designing chromatic multifocal optics and serves as a theoretical foundation for interpretting results

Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs

Integrated model of the vertebrate augmin complex

Accurate segregation of chromosomes is required to maintain genome integrity during cell division. This feat is accomplished by the microtubule-based spindle. To build a spindle rapidly and with high fidelity, cells take advantage of branching microtubule nucleation, which rapidly amplifies microtubules during cell division. Branching microtubule nucleation relies on the hetero-octameric augmin complex, but lack of structure information about augmin has hindered understanding how it promotes branching. In this work, we combine cryo-electron microscopy, protein structural prediction, and visualization of fused bulky tags via negative stain electron microscopy to identify the location and orientation of each subunit within the augmin structure. Evolutionary analysis shows that augmin\u27s structure is highly conserved across eukaryotes, and that augmin contains a previously unidentified microtubule binding site. Thus, our findings provide insight into the mechanism of branching microtubule nucleation

Insights into Translational Termination from the Structure of RF2 Bound to the Ribosome

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release

Electronic energy migration in Microtubules

The repeating arrangement of tubulin dimers confers great mechanical strength to microtubules, which are used as scaffolds for intracellular macromolecular transport in cells and exploited in biohybrid devices. The crystalline order in a microtubule, with lattice constants short enough to allow energy transfer between amino acid chromophores, is similar to synthetic structures designed for light harvesting. After photoexcitation, can these amino acid chromophores transfer excitation energy along the microtubule like a natural or artificial light-harvesting system? Here, we use tryptophan autofluorescence lifetimes to probe energy hopping between aromatic residues in tubulin and microtubules. By studying how the quencher concentration alters tryptophan autofluorescence lifetimes, we demonstrate that electronic energy can diffuse over 6.6 nm in microtubules. We discover that while diffusion lengths are influenced by tubulin polymerization state (free tubulin versus tubulin in the microtubule lattice), they are not significantly altered by the average number of protofilaments (13 versus 14). We also demonstrate that the presence of the anesthetics etomidate and isoflurane reduce exciton diffusion. Energy transport as explained by conventional Förster theory (accommodating for interactions between tryptophan and tyrosine residues) does not sufficiently explain our observations. Our studies indicate that microtubules are, unexpectedly, effective light harvesters

The life of a microtubule

The Minisymposium “The Life of a Microtubule: Birth, Dynamics and Function” highlighted new findings on how microtubules (MTs) are made, how their length and spatial organization is regulated, and finally how they contribute to cellular functions

Mechanisms of Mitotic Spindle Assembly

Life depends on cell proliferation and the accurate segregation of chromosomes, which are mediated by the microtubule (MT)-based mitotic spindle and ~200 essential MT-associated proteins. Yet, a mechanistic understanding of how the mitotic spindle is assembled and achieves chromosome segregation is still missing. This is mostly due to the density of MTs in the spindle, which presumably precludes their direct observation. Recent insight has been gained into the molecular building plan of the metaphase spindle using bulk and single-molecule measurements combined with computational modeling. MT nucleation was uncovered as a key principle of spindle assembly, and mechanistic details about MT nucleation pathways and their coordination are starting to be revealed. Lastly, advances in studying spindle assembly can be applied to address the molecular mechanisms of how the spindle segregates chromosomes