Three-dimensional, tomographic super-resolution fluorescence imaging of serially sectioned thick samples

Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 μm×50 μm×2.5 μm. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.     

Live-cell imaging

It would be hard to argue that live-cell imaging has not changed our view of biology. The past 10 years have seen an explosion of interest in imaging cellular processes, down to the molecular level. There are now many advanced techniques being applied to live cell imaging. However, cellular health is often under appreciated. For many researchers, if the cell at the end of the experiment has not gone into apoptosis or is blebbed beyond recognition, than all is well. This is simply incorrect. There are many factors that need to be considered when performing live-cell imaging in order to maintain cellular health such as: imaging modality, media, temperature, humidity, PH, osmolality, and photon dose. The wavelength of illuminating light, and the total photon dose that the cells are exposed to, comprise two of the most important and controllable parameters of live-cell imaging. The lowest photon dose that achieves a measureable metric for the experimental question should be used, not the dose that produces cover photo quality images. This is paramount to ensure that the cellular processes being investigated are in their in vitro state and not shifted to an alternate pathway due to environmental stress. The timing of the mitosis is an ideal canary in the gold mine, in that any stress induced from the imaging will result in the increased length of mitosis, thus providing a control model for the current imagining conditions.     

3D multicolor super-resolution imaging offers improved accuracy in neuron tracing

The connectivity among neurons holds the key to understanding brain function. Mapping neural connectivity in brain circuits requires imaging techniques with high spatial resolution to facilitate neuron tracing and high molecular specificity to mark different cellular and molecular populations. Here, we tested a three-dimensional (3D), multicolor super-resolution imaging method, stochastic optical reconstruction microscopy (STORM), for tracing neural connectivity using cultured hippocampal neurons obtained from wild-type neonatal rat embryos as a model system. Using a membrane specific labeling approach that improves labeling density compared to cytoplasmic labeling, we imaged neural processes at 44 nm 2D and 116 nm 3D resolution as determined by considering both the localization precision of the fluorescent probes and the Nyquist criterion based on label density. Comparison with confocal images showed that, with the currently achieved resolution, we could distinguish and trace substantially more neuronal processes in the super-resolution images. The accuracy of tracing was further improved by using multicolor super-resolution imaging. The resolution obtained here was largely limited by the label density and not by the localization precision of the fluorescent probes. Therefore, higher image resolution, and thus higher tracing accuracy, can in principle be achieved by further improving the label density.     

Live-cell imaging of cAMP dynamics

Spatial and temporal compartmentalization of cAMP (and its target proteins) is central to the ability of this second messenger to govern cellular activity over timescales ranging from milliseconds to several hours. Recent years have witnessed a burgeoning of methodologies that enable researchers to directly monitor rapid subcellular cAMP dynamics, which are unobtainable by traditional cAMP assays. In this review, we examine cAMP biosensors that are currently available for measuring cAMP at the single-cell level, compare their various operating principles and discuss their applications.     

Live-cell imaging of Aspergillus nidulans autophagy

We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This ‘autophagosome cycle’ gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS^RAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS^RAB7 or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA^RAB5/RabB^RAB5 (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE₂-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO^RAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO^RAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO^RAB1 are dissociable by mutation: rabO^A136D hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.     

Deep and clear optical imaging of thick inhomogeneous samples

Inhomogeneity in thick biological specimens results in poor imaging by light microscopy, which deteriorates as the focal plane moves deeper into the specimen. Here, we have combined selective plane illumination microscopy (SPIM) with wavefront sensor adaptive optics (wao). Our waoSPIM is based on a direct wavefront measure using a Hartmann-Shack wavefront sensor and fluorescent beads as point source emitters. We demonstrate the use of this waoSPIM method to correct distortions in three-dimensional biological imaging and to improve the quality of images from deep within thick inhomogeneous samples.     

Live-cell imaging of vegetative hyphal fusion in Neurospora crassa

The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenk?rper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.     

4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues

Background

Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.

Methodology/Principal Findings

We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.

Conclusions/Significance

Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.     

Live-cell imaging in the era of too many microscopes

At the time of this writing, searching Google Scholar for ‘light-sheet microscopy’ returns almost 8500 results; over three-quarters of which were published in the last 5 years alone. Searching for other advanced imaging methods in the last 5 years yields similar results: ‘super-resolution microscopy’ (>16 000), ‘single-molecule imaging’ (almost 10 000), SPIM (Single Plane Illumination Microscopy, 5000), and ‘lattice light-sheet’ (1300). The explosion of new imaging methods has also produced a dizzying menagerie of acronyms, with over 100 different species of ‘light-sheet’ alone, from SPIM to UM (Ultra microscopy) to SiMView (Simultaneous MultiView) to iSPIM (inclined SPIM, not to be confused with iSPIM, inverted SPIM). How then is the average biologist, without an advanced degree in physics, optics, or computer science supposed to make heads or tails of which method is best suited for their needs? Let us also not forget the plight of the optical physicist, who at best might need help with obtaining healthy samples and keeping them that way, or at worst may not realize the impact their newest technique could have for biologists. This review will not attempt to solve all these problems, but instead highlight some of the most recent, successful mergers between biology and advanced imaging technologies, as well as hopefully provide some guidance for anyone interested in journeying into the world of live-cell imaging.     

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples

C-3-substituted 25-hydroxyvitamin D₃ analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3β-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue–DBP complex. When compared to 25-hydroxyvitamin D₃-DBP, the binding of some antibodies to the analogue–DBP complex of the 25-hydroxyvitamin D₃ derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

Live-cell imaging of rhabdovirus-induced morphological changes in plant nuclear membranes

Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virus-specific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plus-strand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes.     

Live-cell imaging demonstrates rapid cargo exchange between lipid droplets in adipocytes

Lipid droplets form the storage reservoirs for lipids in adipocytes, and their stable appearance suggests a static nature of lipid storage. A stable lipid store, however, may be maintained through the dynamic recycling of lipid cargo between the cytoplasmic compartment and the lipid droplet. In this study, we applied live-cell microscopy to follow intracellular transport steps of fluorescently labeled fatty acids in differentiated 3T3-L1 adipocytes. We demonstrate that intracellular lipids continuously exit and re-enter lipid droplets, and that individual lipid droplets exchange their content on a timescale of minutes. These data demonstrate a surprisingly high rate of intracellular lipid turnover in adipocytes and support the novel concept that lipid storage is achieved by dynamic recycling rather than static retention.     

Constructing complex 3D biological environments from medical imaging using high performance computing

Extracting information about the structure of biological tissue from static image data is a complex task requiring computationally intensive operations. Here, we present how multicore CPUs and GPUs have been utilized to extract information about the shape, size, and path followed by the mammalian oviduct, called the fallopian tube in humans, from histology images, to create a unique but realistic 3D virtual organ. Histology images were processed to identify the individual cross sections and determine the 3D path that the tube follows through the tissue. This information was then related back to the histology images, linking the 2D cross sections with their corresponding 3D position along the oviduct. A series of linear 2D spline cross sections, which were computationally generated for the length of the oviduct, were bound to the 3D path of the tube using a novel particle system technique that provides smooth resolution of self-intersections. This results in a unique 3D model of the oviduct, which is grounded in reality. The GPU is used for the processor intensive operations of image processing and particle physics based simulations, significantly reducing the time required to generate a complete model.     

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.     

Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories

Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.     

Live-cell imaging approaches for the investigation of xenobiotic-induced oxidant stress

BackgroundOxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes.Scope of reviewThe study of events involved in redox toxicology requires methodology capable of detecting transient modifications at relatively low signal strength. This article reviews the advantages of live-cell imaging for redox toxicology studies.Major conclusionsToxicological studies with xenobiotics of supra-physiological reactivity require careful consideration when using fluorogenic sensors in order to avoid potential artifacts and false negatives. Fortunately, experiments conducted for the purpose of validating the use of these sensors in toxicological applications often yield unexpected insights into the mechanisms through which xenobiotic exposure induces oxidant stress.General significanceLive-cell imaging using a new generation of small molecule and genetically encoded fluorophores with excellent sensitivity and specificity affords unprecedented spatiotemporal resolution that is optimal for redox toxicology studies. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.