Simple generation of cleavable labels for multiplexed imaging.

The most common methods for multiplexed immunohistochemistry rely on cyclic procedures, whereby cells or tissues are repeatedly stained, imaged, and regenerated. Here, we present a simple and inexpensive approach for amine-targeted labeling of antibodies using a linker that can be easily cleaved by a mild reducing agent. This method requires only inexpensive and readily-available reagents, and can be carried out without synthetic experience in a simple one-pot reaction.

Simultaneous multicolor fluorescence imaging using PSF splitting.

We present a way to encode more information in fluorescence imaging by splitting the original point spread function (PSF), which offers broadband operation and compatibility with other PSF engineering modalities and existing analysis tools. We demonstrate the approach using the 'Circulator', an add-on that encodes the fluorophore emission band into the PSF, enabling simultaneous multicolor super-resolution and single-molecule microscopy using essentially the full field of view.

Per-pixel unmixing of spectrally overlapping fluorophores using intra-exposure excitation modulation.

Multilabel fluorescence imaging is essential for the visualization of complex systems, though a major challenge is the limited width of the useable spectral window. Here, we present a new method, exNEEMO, that enables per-pixel quantification of spectrally-overlapping fluorophores based on their light-induced dynamics, in a way that is compatible with a very broad range of timescales over which these dynamics may occur. Our approach makes use of intra-exposure modulation of the excitation light to distinguish the different emitters given their reference responses to this modulation.

Evidence of Cytolysin A nanopore incorporation in mammalian cells assessed by a graphical user interface.

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensors allow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology.

Quantitative determination of the full switching cycle of photochromic fluorescent proteins.

In this study, we develop a general analytical model of the photochromism of fluorescent proteins and apply it to spectroscopic measurements performed on six different labels. Our approach provides quantitative explanations for phenomena such as the existence of positive and negative switching, limitations in the photochromism contrast, and the fact that initial switching cycles may differ from subsequent ones. It also allows us to perform the very first measurement of all four isomerization quantum yields involved in the switching process.

Live-Cell SOFI Correlation with SMLM and AFM Imaging.

Standard optical imaging is diffraction-limited and lacks the resolving power to visualize many of the organelles and proteins found within the cell. The advent of super-resolution techniques overcame this barrier, enabling observation of subcellular structures down to tens of nanometers in size; however these techniques require or are typically applied to fixed samples. This raises the question of how well a fixed-cell image represents the system prior to fixation.

Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry.

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4 °C and a few days at 37 °C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate.

Smart genetically-encoded biosensors for the chemical monitoring of living systems.

Genetically-encoded biosensors provide the all-optical and non-invasive visualization of dynamic biochemical events within living systems, which has allowed the discovery of profound new insights. Twenty-five years of biosensor development has steadily improved their performance and has provided us with an ever increasing biosensor repertoire. In this feature article, we present recent advances made in biosensor development and provide a perspective on the future direction of the field.

Modifying PCDH19 levels affects cortical interneuron migration.

PCDH19 is a transmembrane protein and member of the protocadherin family. It is encoded by the X-chromosome and more than 200 mutations have been linked to the neurodevelopmental PCDH-clustering epilepsy (PCDH19-CE) syndrome. A disturbed cell-cell contact that arises when random X-inactivation creates mosaic absence of PCDH19 has been proposed to cause the syndrome.

Spectrally Tunable Förster Resonance Energy Transfer-Based Biosensors Using Organic Dye Grafting.

Biosensors based on Förster resonance energy transfer (FRET) have revolutionized cellular biology by allowing the direct measurement of biochemical processes . Many genetically encoded sensors make use of fluorescent proteins that are limited in spectral versatility and that allow few ways to change the spectral properties once the construct has been created. In this work, we developed genetically encoded FRET biosensors based on the chemigenetic SNAP and HaloTag domains combined with matching organic fluorophores.

Synchronized, Spontaneous, and Oscillatory Detachment of Eukaryotic Cells: A New Tool for Cell Characterization and Identification.

Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label-free methods without (bio)-chemical markers or surface-engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label- and receptor-free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell-detachment with sharply defined time-patterns, a phenomenon unknown from literature.

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 Ca biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca concentration in live cells.

Multilinear Slicing for curve resolution of fluorescence imaging with sequential illumination.

Fluorescence microscopy is an extremely powerful technique that allows to distinguish multiple labels based on their emission color or other properties, such as their photobleaching and fluorescence recovery kinetics. These kinetics are ideally assumed to be mono-exponential in nature, where the time constants intrinsic to each fluorophore can be used to quantify their presence in the sample. However, these time constants also depend on the specifics of the illumination and sample conditions, meaning that identifying the different contributions in a mixture using a single-channel detection may not be straightforward.

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy.

Background: The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy.

Separation of spectrally overlapping fluorophores using intra-exposure excitation modulation.

Multicolor fluorescence imaging is an excellent method for the simultaneous visualization of multiple structures, although it is limited by the available spectral window. More labels can be measured by distinguishing these on properties, such as their fluorescence dynamics, but usually these dynamics must be directly resolvable by the instrument. We propose an approach to distinguish emitters over a much broader range of light-induced dynamics by combining fast modulation of the light source with the detection of the time-integrated fluorescence.

Self-contained and modular structured illumination microscope.

We present a modular implementation of structured illumination microscopy (SIM) that is fast, largely self-contained and that can be added onto existing fluorescence microscopes. Our strategy, which we call HIT-SIM, can theoretically deliver well over 50 super-resolved images per second and is readily compatible with existing acquisition software packages. We provide a full technical package consisting of schematics, a list of components and an alignment scheme that provides detailed specifications and assembly instructions.

Design of experiments for the optimization of SOFI super-resolution microscopy imaging.

Super-resolution optical fluctuation imaging (SOFI) is a well-known super-resolution technique appreciated for its versatility and broad applicability. However, even though an extended theoretical description is available, it is still not fully understood how the interplay between different experimental parameters influences the quality of a SOFI image. We investigated the relationship between five experimental parameters (measurement time, on-time , off-time , probe brightness, and out of focus background) and the quality of the super-resolved images they yielded, expressed as Signal to Noise Ratio (SNR).

Actuation enhances patterning in human neural tube organoids.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids.

Smoothness correction for better SOFI imaging.

Sub-diffraction or super-resolution fluorescence imaging allows the visualization of the cellular morphology and interactions at the nanoscale. Statistical analysis methods such as super-resolution optical fluctuation imaging (SOFI) obtain an improved spatial resolution by analyzing fluorophore blinking but can be perturbed by the presence of non-stationary processes such as photodestruction or fluctuations in the illumination. In this work, we propose to use Whittaker smoothing to remove these smooth signal trends and retain only the information associated to independent blinking of the emitters, thus enhancing the SOFI signals.

Simultaneous readout of multiple FRET pairs using photochromism.

Förster resonant energy transfer (FRET) is a powerful mechanism to probe associations in situ. Simultaneously performing more than one FRET measurement can be challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We present an approach to distinguish overlapping FRET pairs based on the photochromism of the donor fluorophores, even if the involved fluorophores display essentially identical absorption and emission spectra.

Photochromic Fluorophores Enable Imaging of Lowly Expressed Proteins in the Autofluorescent Fungus Candida albicans.

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.

Quantitative Model for Reversibly Photoswitchable Sensors.

Composed of a reversibly photoswitchable unit allosterically linked to a sensing module, reversibly photoswitchable sensors (rs-sensors) represent a new and attractive strategy to quantitatively read-out analyte concentrations. However, their kinetic response to illumination is complex, and much attention is required from the design to the application steps. Here, we exploit a generic kinetic model of rs-sensors which enables us to point to key thermokinetic parameters, such as dissociation constants and kinetic rates for exchange toward the analyte, and cross-sections for photoswitching.