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Oral drug delivery is a preferred method for drug administration because it is economical and convenient to the patient [1]. Uptake from the gut to the blood is mediated by the intestinal barrier, which exhibits some selectivity, i.e., mechanisms in the intestinal barrier define which compounds are absorbed and which are not. The limited understanding of these mechanisms impedes the design and development of new oral drugs. On the horizon: Emerging organ-on-a-chip models of the intestine may provide new insights into structure-function relationships in biological barriers like the gut-blood interface of the intestine. These small ‘artificial organs’ can host biological cells, which mimic the natural behavior of intestinal cells, thus providing a highly controlled platform for fundamental bioresearch and, ultimately, drug-screening [2]. Our group develops and implements optical imaging technologies specifically geared for imaging organ-on-a-chip systems. At the SPIE conference “Optical Methods for Inspection, Characterization, and Imaging of Biomaterials V” we will present the application of two-photon microscopy for imaging of relatively thick 3D printed organ-on-a-chip systems and discuss imaging-challenges related to sample-induced aberrations and scattering. We will also present our ongoing work to implement an adaptive optics-enabled lattice light sheet microscope [3], which (we anticipate) will enable high-resolution imaging inside complex organ-on-a-chip systems by compensating the sample-induced aberrations. [1] P. Viswanathan, Y. Muralidaran, and G. Ragavan, "Challenges in oral drug delivery: a nano-based strategy to overcome", Nanostructures for Oral Medicine, Elsevier (2017) 173-201. [2] R. Zhang and N.B. Larsen, "Stereolithographic hydrogel printing of 3D culture chips with biofunctionalized complex 3D perfusion networks", Lab on a Chip 17.24 (2017) 4273-4282. [3] T. Liu, S. Upadhyayula, et al., "Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms", Science 360.6386 (2018) eaaq1392.

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.

Besides its well-established role in generating COPI vesicles by recruiting coatomer at the Golgi, the small GTPase ARF1 is additionally involved in the formation of anterograde and retrograde tubular carriers at 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.

Summary 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., Graphical Abstract, Highlights • Whole-cell 4Pi single-molecule switching nanoscopy allows 10- to 20-nm 3D resolution • Refined hardware and new data analysis allow imaging of cells as thick as ∼10 μm • Using structure-averaging, the 3D shape of a bacteriophage can be resolved • Wide applicability across diverse research fields is demonstrated, A new super-resolution microscope opens a window deep into cells to image organelles and subcellular structures spanning large volumes.

Stimulated emission depletion (STED) microscopy has been established as an important technique for imaging below the diffraction limit facilitating new discoveries in an array of biological systems. In STED microscopy a “donut-shaped” laser focus is super-imposed onto the diffraction-limited focus of an excitation laser. The dounut-shaped beam suppresses fluorescence in the periphery of the excitation spot, reducing the effective point spread function to a sub-diffraction size. However, the application of multicolor STED microscopy in living cells poses a number of challenges. Here we detail a novel STED system specifically designed for two-color STED applications. Our system employs FPGA-based gated detection and fast beam scanning to reduce pixel dwell time and photobleaching. We demonstrate the instrument’s capability with two-color continuous imaging of intracellular targets below the diffraction limit allowing observation of rare events within live-cells.

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., The intracellular applications of STED microscopy are limited by the availability of dyes. Here the authors develop a two-colour labelling strategy based on SiR and ATTO590 dyes, and apply their strategy to image various subcellular membrane compartments.

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.

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.

When near the focus, the image of a point emitter has little information about the emitter's position along the optical axis. To improve the 3D localization precision, several methods such as astigmatic imaging, double helix point spread functions, and multiple focal plane strategies have been developed to encode the axial position in one or more 2D images of the same emitter. In each of these strategies, a proper model for the 3D microscope point spread function (PSF) is essential to avoid inaccuracies in localization. Generating PSF models from phase retrieved (PR) pupil functions has the advantage that the model images are noise free, can be calculated for any focal plane, and include aberrations specific to the optical system. This representation can also be extremely compact by representing the pupil function as a set of Zernike polynomials. Here we demonstrate the use of PSFs generated from PR pupil functions for 3D single molecule localization based super-resolution imaging using a dual focal plane setup. We show the phase retrieval process, as well as the details of fast PSF calculation by implementation on graphics processing unit hardware. We show a comparison between fitting with ideal, aberration free PSF models and that generated from PR pupil functions using a variety of cellular structures including the ER, microtubules and the membrane proteins STIM1 and ORAI1.

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.