Ultra-High Resolution 3D Imaging of Whole Cells.

Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

Coma aberrations in combined two- and three-dimensional STED nanoscopy.

Stimulated emission depletion (STED) microscopes, like all super-resolution methods, are sensitive to aberrations. Of particular importance are aberrations that affect the quality of the depletion focus, which requires a point of near-zero intensity surrounded by strong illumination. We present analysis, modeling, and experimental measurements that show the effects of coma aberrations on depletion patterns of two-dimensional (2D) and three-dimensional (3D) STED configurations. Specifically, we find that identical coma aberrations create focal shifts in opposite directions in 2D and 3D STED. This phenomenon could affect the precision of microscopic measurements and has ramifications for the efficacy of combined 2D/3D STED systems.

Three dimensional single molecule localization using a phase retrieved pupil function.

Localization-based superresolution imaging is dependent on finding the positions of individual fluorophores in a sample by fitting the observed single-molecule intensity pattern to the microscope point spread function (PSF). For three-dimensional imaging, system-specific aberrations of the optical system can lead to inaccurate localizations when the PSF model does not account for these aberrations. Here we describe the use of phase-retrieved pupil functions to generate a more accurate PSF and therefore more accurate 3D localizations. The complex-valued pupil function contains information about the system-specific aberrations and can thus be used to generate the PSF for arbitrary defocus. Further, it can be modified to include depth dependent aberrations. We describe the phase retrieval process, the method for including depth dependent aberrations, and a fast fitting algorithm using graphics processing units. The superior localization accuracy of the pupil function generated PSF is demonstrated with dual focal plane 3D superresolution imaging of biological structures.

Auto-aligning stimulated emission depletion microscope using adaptive optics.

Stimulated emission depletion (STED) microscopy provides diffraction-unlimited resolution in fluorescence microscopy. Imaging at the nanoscale, however, requires precise alignment of the depletion and excitation laser foci of the STED microscope. We demonstrate here that adaptive optics can be implemented to automatically align STED and confocal images with a precision of 4.3 ± 2.3 nm.

Quantitative pupil analysis in stimulated emission depletion microscopy using phase retrieval.

The resolution attainable with stimulated emission depletion (STED) microscopy greatly depends on the quality of the STED laser focus. So far, visual inspection of a measured STED focus has been the only convenient means of gauging the source of aberrations. Here we describe a method, requiring no instrument modifications, for obtaining an equivalent to the complex pupil function at the back aperture of the objective and show that it provides quantitative information about aberration sources (including aberrations induced by the objective or sample). We show the accuracy of this field representation to be sufficient for reconstructing the STED focus in three dimensions and determining corrective steps.

Pitfalls and opportunities in quantitative fluorescence-based nanomedicine studies - A commentary.

Fluorescence-based techniques are prevalent in studies of nanomedicine-targeting to cells and tissues. However, fluorescence-based studies are rarely quantitative, thus prohibiting direct comparisons of nanomedicine-performance across studies. With this Commentary, we aim to provoke critical thinking about experimental design by treating some often-overlooked pitfalls in 'quantitative' fluorescence-based experimentation. Focusing on fluorescence-labeled nanoparticles, we cover mechanisms like solvent-interactions and fluorophore-dissociation, which disqualify the assumption that 'a higher fluorescence readout' translates directly to 'a better targeting efficacy'. With departure in recent literature, we propose guidelines for circumventing these pitfalls in studies of tissue-accumulation and cell-uptake, thus covering fluorescence-based techniques like bulk solution fluorescence measurements, fluorescence microscopy, flow cytometry, and infrared fluorescence imaging. With this, we hope to lay a foundation for more 'quantitative thinking' during experimental design, enabling (for example) the estimation and reporting of actual numbers of fluorescent nanoparticles accumulated in cells and organs.

Dual-Material 3D-Printed Intestinal Model Devices with Integrated Villi-like Scaffolds.

In vitro small intestinal models aim to mimic the in vivo intestinal function and structure, including the villi architecture of the native tissue. Accurate models in a scalable format are in great demand to advance, for example, the development of orally administered pharmaceutical products. Widely used planar intestinal cell monolayers for compound screening applications fail to recapitulate the three-dimensional (3D) microstructural characteristics of the intestinal villi arrays. This study employs stereolithographic 3D printing to manufacture biocompatible hydrogel-based scaffolds with villi-like micropillar arrays of tunable dimensions in poly(ethylene glycol) diacrylates (PEGDAs). The resulting 3D-printed microstructures are demonstrated to support a month-long culture and induce apicobasal polarization of Caco-2 epithelial cell layers along the villus axis, similar to the native intestinal microenvironment. Transport analysis requires confinement of compound transport to the epithelial cell layer within a compound diffusion-closed reservoir compartment. We meet this challenge by sequential printing of PEGDAs of different molecular weights into a monolithic device, where a diffusion-open villus-structured hydrogel bottom supports the cell culture and mass transport within the confines of a diffusion-closed solid wall. As a functional demonstrator of this scalable dual-material 3D micromanufacturing technology, we show that Caco-2 cells seeded in villi-wells form a tight epithelial barrier covering the villi-like micropillars and that compound-induced challenges to the barrier integrity can be monitored by standard high-throughput analysis tools (fluorescent tracer diffusion and transepithelial electrical resistance).

Novel ecto-tagged integrins reveal their trafficking in live cells.

Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Integrin function is dynamically modulated by endo-exocytic trafficking, however, major mysteries remain about where, when, and how this occurs in living cells. To address this, here we report the generation of functional recombinant ß1 integrins with traceable tags inserted in an extracellular loop. We demonstrate that these 'ecto-tagged' integrins are cell-surface expressed, localize to adhesions, exhibit normal integrin activation, and restore adhesion in ß1 integrin knockout fibroblasts. Importantly, ß1 integrins containing an extracellular pH-sensitive pHluorin tag allow direct visualization of integrin exocytosis in live cells and revealed targeted delivery of integrin vesicles to focal adhesions. Further, using ß1 integrins containing a HaloTag in combination with membrane-permeant and -impermeant Halo dyes allows imaging of integrin endocytosis and recycling. Thus, ecto-tagged integrins provide novel powerful tools to characterize integrin function and trafficking.Integrins are cell-surface adhesion receptors that are modulated by endo-exocytic trafficking, but existing tools to study this process can interfere with function. Here the authors develop ß1 integrins carrying traceable tags in the extracellular domain; a pH-sensitive pHlourin tag or a HaloTag to facilitate dye attachment.

A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.

Two-colour live-cell nanoscale imaging of intracellular targets.

Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.