Integrated multimodality microscope for accurate and efficient target-guided cryo-lamellae preparation.

Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.

Molecular-scale axial localization by repetitive optical selective exposure.

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures.

Author Correction: Molecular resolution imaging by repetitive optical selective exposure.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular resolution imaging by repetitive optical selective exposure.

We introduce an interferometric single-molecule localization method for super-resolution fluorescence microscopy. Fluorescence molecules are located by the intensities of multiple excitation patterns of an interference fringe, providing around a twofold improvement in the localization precision compared with the conventional imaging with the same photon budget. We demonstrate this technique by resolving nanostructures down to 5 nm in size over a large 25 × 25 µm2 field of view.

Interferometrical single-molecule localization based on dynamic PSF engineering: publisher's note.

This publisher's note contains a correction to Opt. Lett.47, 1770 (2022)10.1364/OL.453113.

Interferometrical single-molecule localization based on dynamic PSF engineering.

We present a method for interferometric single-molecule localization based on dynamic point spread function (PSF) engineering. By using two galvo mirrors, a hexagonal PSF is constructed and the fluorescent signal under different illumination patterns could be acquired simultaneously. This method was evaluated using simulation, fluorescent nanosphere imaging, and single-molecule imaging. The study indicates a twofold improvement in localization precision while maintaining the same photon budget. This strategy, we believe, is a cost-effective way to increase the resolution of single-molecule localization microscopy.

High-density 3D single molecular analysis based on compressed sensing.

Single molecule fitting-based superresolution microscopy achieves sub-diffraction-limit image resolution but suffers from a need for long acquisition times to gather enough molecules. Several methods have recently been developed that analyze high molecule density images but most are only applicable to two dimensions. In this study, we implemented a high-density superresolution localization algorithm based on compressed sensing and a biplane approach that provides three-dimensional information about molecules, achieving super-resolution imaging at higher molecule densities than those achieved using the conventional single molecule fitting method.

CAMSAP2 and CAMSAP3 localize at microtubule intersections to regulate the spatial distribution of microtubules.

Microtubule networks support many cellular processes and have a highly ordered architecture. However, due to the limited axial resolution of conventional light microscopy, the structural features of these networks cannot be resolved in three-dimensional (3D) space. Here, we use customized ultra-high resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells. We find that the microtubule minus-ends associated protein CAMSAPs localize at a portion of microtubule intersections. Further investigation shows that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance. We find that CAMSAPs recognize microtubule defects, which are often associated with microtubule intersections, and then recruit katanin to remove the damaged microtubules. Therefore, the CAMSAP-katanin complex is a regulating module for the distance between microtubules. Taken together, our results characterize the architecture of the cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.

Tetra-color superresolution microscopy based on excitation spectral demixing.

Multicolor imaging allows protein colocalizations and organelle interactions to be studied in biological research, which is especially important for single-molecule localization microscopy (SMLM). Here, we propose a multicolor method called excitation-resolved stochastic optical reconstruction microscopy (ExR-STORM). The method, which is based on the excitation spectrum of fluorescent dyes, successfully separated four spectrally very close far-red organic fluorophores utilizing three excitation lasers with cross-talk of less than 3%. Dyes that are only 5 nm apart in the emission spectrum were resolved, resulting in negligible chromatic aberrations. This method was extended to three-dimensional (3D) imaging by combining the astigmatic method, providing a powerful tool for resolving 3D morphologies at the nanoscale.

CCDC176 stabilizes microtubule doublets 1 and 9 to ensure proper sperm movement.

The molecular mechanism underlying asymmetric axonemal complexes in sperm flagella is still largely unknown. Here, we showed that the knockout of the coiled-coil domain-containing 176 (CCDC176) in mice led to male infertility due to decreased sperm motility. Ccdc176 knockout specifically destabilized microtubule doublets (MTDs) 1 and 9 during sperm maturation in the corpus epididymis. Single-sperm immunofluorescence showed that most CCDC176 was distributed along the axoneme, and further super-resolution imaging revealed that CCDC176 is asymmetrically localized in the sperm axoneme. CCDC176 could cooperate with microtubule and radial spoke proteins to stabilize MTDs 1 and 9, and its knockout results in the destabilization of some proteins in sperm flagella. Furthermore, as predicted by the sperm multibody dynamics (MBD) model, we found that MTDs 1 and 9 jutted out from the sperm flagellum annulus region in Ccdc176-/- spermatozoa, and these flagellar defects alter sperm flagellar beat patterns and swimming paths, potentially owing to the reduction and disequilibration of bending torque on the central pair. These results demonstrate that CCDC176 specifically stabilizes MTDs 1 and 9 in the sperm flagellum to ensure proper sperm movement for fertilization.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

Recent progress on single-molecule localization microscopy.

Super-resolution imaging based on single-molecule localization has been developed for more than a decade. These techniques can break through diffraction limit of fluorescent microscopy and initially improve the resolution by an order of magnitude to ~20 nm, by introducing photoactivatable/photoswitching probes and centroid fitting method. As the demand of biological research, the localization precision of single-molecules was further improved by several state-of-the-art methods in the past several years. This review focuses on the latest developed techniques which have greatly improved the performance of single-molecule localization microscopy, from measurement principle to hardware design. These methods are essential for the study of nanostructures and biomacromolecule dynamics inside of cells.

Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors.

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo, yet the underlying mechanisms remain to be explored. We showed here that the spatial distribution of interleukin-4 (IL4) in invariant natural killer T (iNKT) cells regulated crosstalk between iNKT cells and dendritic cells (DCs) and controlled iNKT cell-mediated T-helper type 1 (Th1) responses. The persistent polarization of IL4 induced by strong lipid antigens, that is, α-galactosylceramide (αGC), caused IL4 accumulation at the immunological synapse (IS), which promoted the activation of the IL4R-STAT6 (signal transducer and activator of transcription 6) pathway and production of IL12 in DCs, which enhanced interferon-γ (IFNγ) production in iNKT cells. Conversely, the nonpolarized secretion of IL4 induced by Th2 lipid antigens with a short or unsaturated chain was incapable of enhancing this iNKT cell-DC crosstalk and thus shifted the immune response to a Th2-type response. The nonpolarized secretion of IL4 in response to Th2 lipid antigens was caused by the degradation of Cdc42 in iNKT cells. Moreover, reduced Cdc42 expression was observed in tumor-infiltrating iNKT cells, which impaired IL4 polarization and disturbed iNKT cell-DC crosstalk in tumors.

Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy.

Remarkable progress in correlative light and electron cryo-microscopy (cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope (cryo-FM). Here, we describe an ultra-stable super-resolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than 0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system (average single molecule localization accuracy of ∼13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy (SMLM) and cryogenic super-resolution correlative light and electron microscopy (csCLEM).

Three-dimensional super-resolution protein localization correlated with vitrified cellular context.

We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

Ultrafast, accurate, and robust localization of anisotropic dipoles.

The resolution of single molecule localization imaging techniques largely depends on the precision of localization algorithms. However, the commonly used Gaussian function is not appropriate for anisotropic dipoles because it is not the true point spread function. We derived the theoretical point spread function of tilted dipoles with restricted mobility and developed an algorithm based on an artificial neural network for estimating the localization, orientation and mobility of individual dipoles. Compared with fitting-based methods, our algorithm demonstrated ultrafast speed and higher accuracy, reduced sensitivity to defocusing, strong robustness and adaptability, making it an optimal choice for both two-dimensional and three-dimensional super-resolution imaging analysis.