Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching.

We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.

Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA.

Sporulation in the bacterium Bacillus subtilis is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σF, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σF. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σF is activated.

Incoherent structured illumination improves optical sectioning and contrast in multiphoton super-resolution microscopy.

Three-dimensional super-resolution imaging in thick, semi-transparent biological specimens is hindered by light scattering, which increases background and degrades both contrast and optical sectioning. We describe a simple method that mitigates these issues, improving image quality in our recently developed two-photon instant structured illumination microscope without requiring any hardware modifications to the instrument. By exciting the specimen with three laterally-structured, phase-shifted illumination patterns and post-processing the resulting images, we digitally remove both scattered and out-of-focus emissions that would otherwise contaminate our raw data. We demonstrate the improved performance of our approach in biological samples, including pollen grains, primary mouse aortic endothelial cells cultured in a three-dimensional collagen matrix and live tumor-like cell spheroids.

Actin-dependent clustering of insulin receptors in membrane microdomains.

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging. Our analyses indicate that binding of insulin to IR on these cells is multivalent, involving at least two insulin molecules per IR as labeling concentrations approach 1µM. Insulin binding also reduces lateral diffusion of IR and the size of membrane compartments accessed by IR. For IR that have not bound insulin, lateral diffusion of IR and the size of membrane compartments accessed by IR increase after disrupting actin filaments or depleting membrane cholesterol. However, clustering of insulin-occupied IR is reduced only by disrupting actin filaments or by fixing cells with paraformaldehyde prior to exposure to insulin, but not by depleting membrane cholesterol. Thus, it appears that, although restriction of IR lateral diffusion on these cells is sensitive to both actin filament dynamics and membrane cholesterol content, clustering of insulin-occupied IR primarily involves an actin-dependent mechanism.

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains.

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM).

Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, four-dimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning- methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online.

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy.

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence.

Untwisting the Caenorhabditis elegans embryo.

The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis.

Faster fluorescence microscopy: advances in high speed biological imaging.

The past decade has seen explosive growth in new high speed imaging methods. These can broadly be classified as either point-scanning (which offer better depth penetration) or parallelized systems (which offer higher speed). We discuss each class generally, and cover specific advances in diffraction-limited microscopes (laser-scanning confocal, spinning-disk, and light-sheet) and superresolution microscopes (single-molecule imaging, stimulated emission-depletion, and structured illumination). A theme of our review is that there is no free lunch: each technique has strengths and weaknesses, and an advance in speed usually comes at the expense of either spatial resolution or depth penetration.

Luteinizing hormone receptors are confined in mesoscale plasma membrane microdomains throughout recovery from receptor desensitization.

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80-160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.

Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 µm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy.

Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.

Insulin receptors and downstream substrates associate with membrane microdomains after treatment with insulin or chromium(III) picolinate.

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)(3)). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 µM Cr(pic)(3). These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)(3) but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)(3) did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)(3) likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)(3) involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)(3) are due to changes in membrane lipid order rather than to direct interactions with IR.

The anti-diabetic bis(maltolato)oxovanadium(IV) decreases lipid order while increasing insulin receptor localization in membrane microdomains.

The effects of treatment with bis(maltolato)oxovanadium(IV) (BMOV) on protein localization in membrane microdomains were investigated by comparing the effects of insulin and treatment with BMOV on the lateral motions and compartmentalization of individual insulin receptors (IR). In addition, effects of insulin and BMOV on the association of IR, phosphorylated IR (pIR) and phosphorylated insulin receptor substrate-1 (pIRS-1) with chemically-isolated plasma membrane microdomains on rat basophilic leukemia (RBL-2H3) cells were evaluated. Single particle tracking experiments indicate that individual quantum dot-labeled IR on RBL-2H3 cells exhibit relatively unrestricted lateral diffusion of approximately 1 × 10(-10) cm(2) s(-1) and are confined in approximately 475 nm diameter cell-surface membrane compartments. After treating of RBL-2H3 cells with 10 µM BMOV, IR lateral diffusion and the size of IR-containing membrane compartments is significantly reduced to 6 × 10(-11) cm(2) s(-1) and approximately 400 nm, respectively. BMOV treatment also increases the association of IR with low-density, detergent-resistant membrane fragments isolated using isopycnic sucrose-gradient centrifugation from 2.4% for untreated cells to 25.8% for cells treated with 10 µM BMOV. Additionally, confocal fluorescence microscopic imaging of live RBL-2H3 cells labeled with the phase sensitive aminonaphthylethenylpyridinium-based dye, Di-4-ANEPPDHQ, indicates that BMOV treatment, but not insulin treatment, decreases cell-surface plasma membrane lipid order while fluorescence correlation spectroscopy measurements suggest that BMOV treatment does not affect IR surface-density or insulin binding affinity. Finally, model studies using microemulsions of cetyltrimethylammonium bromide (CTAB) micelles and (1)H NMR spectroscopy show that an oxidized form of BMOV readily localizes near the CTAB head-groups at the lipid-water interface. These observations were supported by IR spectroscopic studies using microemulsions of CTAB reverse micelles showing that both BMOV and oxidized BMOV are associated with the water pool. This conclusion is based on changes in (1)H NMR chemical shifts observed for the complex, oxidized BMOV. Moreover, these shifts appeared to be informative about the location of the complex. No differences were observed in the OD absorption peak positions for the CTAB reverse micelles prepared in the presence and absence of BMOV, oxidized BMOV or maltol. Combined, these results suggest that activation of IR signaling by both insulin and BMOV treatment involves increased association of IR with specialized, nanoscale membrane microdomains. The observed insulin-like activity of BMOV or decomposition products of BMOV may be due to changes in cell-surface membrane lipid order rather than due to direct interactions with IR.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells.

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K(D) of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K(D) of 0.14 nM and for the high-affinity insulin binding sites with K(D) of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min(-1) and 0.013 min(-1), respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.