Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification.

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies.

The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.

Video-rate multi-color structured illumination microscopy with simultaneous real-time reconstruction.

Super-resolved structured illumination microscopy (SR-SIM) is among the fastest fluorescence microscopy techniques capable of surpassing the optical diffraction limit. Current custom-build instruments are able to deliver two-fold resolution enhancement with high acquisition speed. SR-SIM is usually a two-step process, with raw-data acquisition and subsequent, time-consuming post-processing for image reconstruction. In contrast, wide-field and (multi-spot) confocal techniques produce high-resolution images instantly. Such immediacy is also possible with SR-SIM, by tight integration of a video-rate capable SIM with fast reconstruction software. Here we present instant SR-SIM by VIGOR (Video-rate Immediate GPU-accelerated Open-Source Reconstruction). We demonstrate multi-color SR-SIM at video frame-rates, with less than 250 ms delay between measurement and reconstructed image display. This is achieved by modifying and extending high-speed SR-SIM image acquisition with a new, GPU-enhanced, network-enabled image-reconstruction software. We demonstrate high-speed surveying of biological samples in multiple colors and live imaging of moving mitochondria as an example of intracellular dynamics.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM.

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.

Primary rat LSECs preserve their characteristic phenotype after cryopreservation.

Liver disease is a leading cause of morbidity and mortality worldwide. Recently, the liver non-parenchymal cells have gained increasing attention for their potential role in the development of liver disease. Liver sinusoidal endothelial cells (LSECs), a specialized type of endothelial cells that have unique morphology and function, play a fundamental role in maintaining liver homeostasis. Current protocols for LSEC isolation and cultivation rely on freshly isolated cells which can only be maintained differentiated in culture for a few days. This creates a limitation in the use of LSECs for research and a need for a consistent and reliable source of these cells. To date, no LSEC cryopreservation protocols have been reported that enable LSECs to retain their functional and morphological characteristics upon thawing and culturing. Here, we report a protocol to cryopreserve rat LSECs that, upon thawing, maintain full LSEC-signature features: fenestrations, scavenger receptor expression and endocytic function on par with freshly isolated cells. We have confirmed these features by a combination of biochemical and functional techniques, and super-resolution microscopy. Our findings offer a means to standardize research using LSECs, opening the prospects for designing pharmacological strategies for various liver diseases, and considering LSECs as a therapeutic target.

A dystroglycan mutation (p.Cys667Phe) associated to muscle-eye-brain disease with multicystic leucodystrophy results in ER-retention of the mutant protein.

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α-DG and the transmembrane ß-DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α-DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O-mannosyl glycans and in the O-mannosylation pathway of α-DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α-DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the ß-DG ectodomain has been identified in a patient affected by muscle-eye-brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α-DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high-resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small-angle X-ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the ß-DG ectodomain overall folding, resulting in disulfide-associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum-retention, underlying dystroglycanopathy.

Comment on "Magnetic-field-enabled resolution enhancement in super-resolution imaging" by M. Zhang et al., Phys. Chem. Chem. Phys., 2015, 17, 6722-6727.

Certain fluorophores, in particular those that can undergo photoinduced radical pair reactions are known to exhibit a magnetic field dependent fluorescence summarized in the term magnetic field effect (MFE). We tried to reproduce experiments that reported magnetic field enhanced fluorescence for commonly used organic dyes with a high quantum yield suitable for single molecule localization microscopy. We find that the enhanced fluorescence is due to fluorescence reflected by the magnet's surface rather than MFE.

Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ.

Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses.

Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations.

Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.

Helium Ion Microscopy Visualizes Lipid Nanodomains in Mammalian Cells.

Cell membranes are composed of 2D bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, direct visualization of cell membrane nanodomains by helium ion microscopy (HIM) is presented. It is shown that HIM is capable to image biological specimens without any conductive coating and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy, and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ≈ 15 nm.

Nanoparticles as Nonfluorescent Analogues of Fluorophores for Optical Nanoscopy.

Optical microscopy modalities that achieve spatial resolution beyond the resolution limit have opened up new opportunities in the biomedical sciences to reveal the structure and kinetics of biological processes on the nanoscale. These methods are, however, mostly restricted to fluorescence as contrast mechanism, which limits the ultimate spatial resolution and observation time that can be achieved by photobleaching of the fluorescent probes. Here, we demonstrate that Raman scattering provides a valuable contrast mechanism for optical nanoscopy in the form of super-resolution structured illumination microscopy. We find that nanotags, i.e., gold and silver nanoparticles that are capable of surface-enhanced Raman scattering (SERS), can be imaged with a spatial resolution beyond the diffraction limit in four dimensions alongside and with similar excitation power as fluorescent probes. The highly polarized nature of super-resolution structured illumination microscopy renders these nanotags elliptical in the reconstructed super-resolved images, which enables us to determine their orientation within the sample. The robustness of nanotags against photobleaching allows us to image these particles for unlimited periods of time. We demonstrate this by imaging isolated nanotags in a dense layer of fluorophores, as well as on the surface of and after internalization by osteosarcoma cells, always in the presence of fluorescent probes. Our results show that SERS nanotags have the potential to become highly multiplexed and chemically sensitive optical probes for optical nanoscopy that can replace fluorophores in applications where fluorescence photobleaching is prohibitive for following the evolution of biological processes for extended times.

Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.

Liver sinusoidal endothelial cells (LSEC) are an important class of endothelial cells facilitating the translocation of lipoproteins and small molecules between the liver and blood. A number of clinical conditions, especially metabolic and aging-related disorders, are implicated by improper function of LSECs. Despite their importance, research into these cells is limited because the primary ultrastructures involved in their function are transcellular pores, called fenestrations, with diameters in a size range between 50-200 nm, i.e. well below the optical diffraction limit. Here, we show that we are able to resolve fenestrations with a spatial resolution of ∼20 nm by direct stochastic optical reconstruction microscopy (dSTORM). The cellular plasma membrane was labeled at high fluorophore density with CellMask Deep Red and imaged using a reducing buffer system. We compare the higher degree of structural detail that dSTORM provides to results obtained by 3D structured illumination microscopy (3D-SIM). Our results open up a path to image these physiologically important cells in vitro using highly resolving localization microscopy techniques that could be implemented on non-specialized fluorescence microscopes, enabling their investigation in most biomedical laboratories without the need for electron microscopy.