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.

Tetrahedral serial multiview microscopy and image fusion for improved resolution and extent in stained zebrafish embryos.

BACKGROUND: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware. RESULTS: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be created from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent. CONCLUSION: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.

Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Leptin stimulates synaptogenesis in hippocampal neurons via KLF4 and SOCS3 inhibition of STAT3 signaling.

Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood. In this study, we reveal two novel targets of leptin in developing hippocampal neurons and address their role in synaptogenesis. First target is Kruppel-Like Factor 4 (KLF4), which we identified using a genome-wide target analysis strategy. We show that leptin upregulates KLF4 in hippocampal neurons and that leptin signaling is important for KLF4 expression in vivo. Furthermore, KLF4 is required for leptin-induced synaptogenesis, as shKLF4 blocks and upregulation of KLF4 phenocopies it. We go on to show that KLF4 requires its signal transducer and activator of transcription 3 (STAT3) binding site and thus potentially blocks STAT3 activity to induce synaptogenesis. Second, we show that leptin increases the expression of suppressor of cytokine signaling 3 (SOCS3), another well-known inhibitor of STAT3, in developing hippocampal neurons. SOCS3 is also required for leptin-induced synaptogenesis and sufficient to stimulate it alone. Finally, we show that constitutively active STAT3 blocks the effects of leptin on spine formation, while the targeted knockdown of STAT3 is sufficient to induce it. Overall, our data demonstrate that leptin increases the expression of both KLF4 and SOCS3, inhibiting the activity of STAT3 in the hippocampal neurons and resulting in the enhancement of glutamatergic synaptogenesis during neonatal development.

Leptin-induced spine formation requires TrpC channels and the CaM kinase cascade in the hippocampus.

Leptin is a critical neurotrophic factor for the development of neuronal pathways and synaptogenesis in the hypothalamus. Leptin receptors are also found in other brain regions, including the hippocampus, and a postnatal surge in leptin correlates with a time of rapid growth of dendritic spines and synapses in the hippocampus. Leptin is critical for normal hippocampal dendritic spine formation as db/db mice, which lack normal leptin receptor signaling, have a reduced number of dendritic spines in vivo. Leptin also positively influences hippocampal behaviors, such as cognition, anxiety, and depression, which are critically dependent on dendritic spine number. What is not known are the signaling mechanisms by which leptin initiates spine formation. Here we show leptin induces the formation of dendritic protrusions (thin headless, stubby and mushroom shaped spines), through trafficking and activation of TrpC channels in cultured hippocampal neurons. Leptin-activation of the TrpC current is dose dependent and blocked by targeted knockdown of the leptin receptor. The nonselective TrpC channel inhibitors SKF96365 and 2-APB or targeted knockdown of TrpC1 or 3, but not TrpC5, channels also eliminate the leptin-induced current. Leptin stimulates the phosphorylation of CaMKIγ and ß-Pix within 5 min and their activation is required for leptin-induced trafficking of TrpC1 subunits to the membrane. Furthermore, we show that CaMKIγ, CaMKK, ß-Pix, Rac1, and TrpC1/3 channels are all required for both the leptin-sensitive current and leptin-induced spine formation. These results elucidate a critical pathway underlying leptin's induction of dendritic morphological changes that initiate spine and excitatory synapse formation.

Using FPbase: The Fluorescent Protein Database.

FPbase is a database of fluorescent proteins and their characteristics and a set of online tools that facilitate searching the database and performing experiments with fluorescent probes. This chapter serves as a general reference for using and searching the database and a guide to some of the more commonly used tools including the spectra viewer, custom microscope pages, and FRET calculator. Important caveats when evaluating the data are also discussed.

The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets.

Lipid droplets (LDs) are crucial organelles for energy storage and lipid homeostasis. Autophagy of LDs is an important pathway for their catabolism, but the molecular mechanisms mediating LD degradation by selective autophagy (lipophagy) are unknown. Here we identify spartin as a receptor localizing to LDs and interacting with core autophagy machinery, and we show that spartin is required to deliver LDs to lysosomes for triglyceride mobilization. Mutations in SPART (encoding spartin) lead to Troyer syndrome, a form of complex hereditary spastic paraplegia1. Interfering with spartin function in cultured human neurons or murine brain neurons leads to LD and triglyceride accumulation. Our identification of spartin as a lipophagy receptor, thus, suggests that impaired LD turnover contributes to Troyer syndrome development.

Multimodal control of dendritic cell functions by nociceptors.

It is known that interactions between nociceptors and dendritic cells (DCs) can modulate immune responses in barrier tissues. However, our understanding of the underlying communication frameworks remains rudimentary. Here, we show that nociceptors control DCs in three molecularly distinct ways. First, nociceptors release the calcitonin gene-related peptide that imparts a distinct transcriptional profile on steady-state DCs characterized by expression of pro-interleukin-1ß and other genes implicated in DC sentinel functions. Second, nociceptor activation induces contact-dependent calcium fluxes and membrane depolarization in DCs and enhances their production of proinflammatory cytokines when stimulated. Finally, nociceptor-derived chemokine CCL2 contributes to the orchestration of DC-dependent local inflammation and the induction of adaptive responses against skin-acquired antigens. Thus, the combined actions of nociceptor-derived chemokines, neuropeptides, and electrical activity fine-tune DC responses in barrier tissues.

Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo.

Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal Days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.

Rapid image deconvolution and multiview fusion for optical microscopy.

The contrast and resolution of images obtained with optical microscopes can be improved by deconvolution and computational fusion of multiple views of the same sample, but these methods are computationally expensive for large datasets. Here we describe theoretical and practical advances in algorithm and software design that result in image processing times that are tenfold to several thousand fold faster than with previous methods. First, we show that an 'unmatched back projector' accelerates deconvolution relative to the classic Richardson-Lucy algorithm by at least tenfold. Second, three-dimensional image-based registration with a graphics processing unit enhances processing speed 10- to 100-fold over CPU processing. Third, deep learning can provide further acceleration, particularly for deconvolution with spatially varying point spread functions. We illustrate our methods from the subcellular to millimeter spatial scale on diverse samples, including single cells, embryos and cleared tissue. Finally, we show performance enhancement on recently developed microscopes that have improved spatial resolution, including dual-view cleared-tissue light-sheet microscopes and reflective lattice light-sheet microscopes.

LDAF1 and Seipin Form a Lipid Droplet Assembly Complex.

Lipid droplets (LDs) originate from the endoplasmic reticulum (ER) to store triacylglycerol (TG) and cholesterol esters. The ER protein seipin was shown to localize to ER-LD contacts soon after LDs form, but what determines the sites of initial LD biogenesis in the ER is unknown. Here, we identify TMEM159, now re-named lipid droplet assembly factor 1 (LDAF1), as an interaction partner of seipin. Together, LDAF1 and seipin form an ∼600 kDa oligomeric complex that copurifies with TG. LDs form at LDAF1-seipin complexes, and re-localization of LDAF1 to the plasma membrane co-recruits seipin and redirects LD formation to these sites. Once LDs form, LDAF1 dissociates from seipin and moves to the LD surface. In the absence of LDAF1, LDs form only at significantly higher cellular TG concentrations. Our data suggest that the LDAF1-seipin complex is the core protein machinery that facilitates LD biogenesis and determines the sites of their formation in the ER.

Age-dependent effects of environmental enrichment on spatial reference memory in male mice.

Although environmental enrichment has been shown to improve various types of memory in young and aging mice, no study has directly compared the degree to which enrichment improves memory at different ages throughout the lifespan in male mice. Therefore, the present study investigated the effects of long-term continuous enrichment in young (3 months), middle-aged (15 months), and aged (21 months) male C57BL/6 mice. Spatial reference memory was tested in the Morris water maze. Results demonstrate that 24h/day environmental enrichment for approximately 6 weeks significantly improved spatial memory in the Morris water maze in aged males, but not in young or middle-aged males. These data also indicate that 24h exposure to complex enriched housing conditions increases the magnitude of enrichment-induced improvements in memory among aged mice relative to those previously reported by this lab and others.

Navigating challenges in the application of superresolution microscopy.

In 2014, the Nobel Prize in Chemistry was awarded to three scientists who have made groundbreaking contributions to the field of superresolution (SR) microscopy (SRM). The first commercial SR microscope came to market a decade earlier, and many other commercial options have followed. As commercialization has lowered the barrier to using SRM and the awarding of the Nobel Prize has drawn attention to these methods, biologists have begun adopting SRM to address a wide range of questions in many types of specimens. There is no shortage of reviews on the fundamental principles of SRM and the remarkable achievements made with these methods. We approach SRM from another direction: we focus on the current practical limitations and compromises that must be made when designing an SRM experiment. We provide information and resources to help biologists navigate through common pitfalls in SRM specimen preparation and optimization of image acquisition as well as errors and artifacts that may compromise the reproducibility of SRM data.

Polo-like kinase-dependent phosphorylation of the synaptonemal complex protein SYP-4 regulates double-strand break formation through a negative feedback loop.

The synaptonemal complex (SC) is an ultrastructurally conserved proteinaceous structure that holds homologous chromosomes together and is required for the stabilization of pairing interactions and the completion of crossover (CO) formation between homologs during meiosis I. Here, we identify a novel role for a central region component of the SC, SYP-4, in negatively regulating formation of recombination-initiating double-strand breaks (DSBs) via a feedback loop triggered by crossover designation in C. elegans. We found that SYP-4 is phosphorylated dependent on Polo-like kinases PLK-1/2. SYP-4 phosphorylation depends on DSB formation and crossover designation, is required for stabilizing the SC in pachytene by switching the central region of the SC from a more dynamic to a less dynamic state, and negatively regulates DSB formation. We propose a model in which Polo-like kinases recognize crossover designation and phosphorylate SYP-4 thereby stabilizing the SC and making chromosomes less permissive for further DSB formation.

Superresolution microscopy of the ß-carboxysome reveals a homogeneous matrix.

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the ß-carboxysome.

Multifocus structured illumination microscopy for fast volumetric super-resolution imaging.

We here report for the first time the synergistic implementation of structured illumination microscopy (SIM) and multifocus microscopy (MFM). This imaging modality is designed to alleviate the problem of insufficient volumetric acquisition speed in super-resolution biological imaging. SIM is a wide-field super-resolution technique that allows imaging with visible light beyond the classical diffraction limit. Employing multifocus diffractive optics we obtain simultaneous wide-field 3D imaging capability in the SIM acquisition sequence, improving volumetric acquisition speed by an order of magnitude. Imaging performance is demonstrated on biological specimens.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.

Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.

Assessing camera performance for quantitative microscopy.

Charge-coupled device and, increasingly, scientific complementary metal oxide semiconductor cameras are the most common digital detectors used for quantitative microscopy applications. Manufacturers provide technical specification data on the average or expected performance characteristics for each model of camera. However, the performance of individual cameras may vary, and many of the characteristics that are important for quantitation can be easily measured. Though it may seem obvious, it is important to remember that the digitized image you collect is merely a representation of the sample itself--and no camera can capture a perfect representation of an optical image. A clear understanding and characterization of the sources of noise and imprecision in your camera are important for rigorous quantitative analysis of digital images. In this chapter, we review the camera performance characteristics that are most critical for generating accurate and precise quantitative data and provide a step-by-step protocol for measuring these characteristics in your camera.