Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.

We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.

Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders.

With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches.

Absolute Arrangement of Subunits in Cytoskeletal Septin Filaments in Cells Measured by Fluorescence Microscopy.

We resolved the organization of subunits in cytoskeletal polymers in cells by light microscopy. Septin GTPases form linear complexes of about 32 nm length that polymerize into filaments. We visualized both termini of septin complexes by single molecule microscopy in vitro. Complexes appeared as 32 nm spaced localization pairs, and filaments appeared as stretches of equidistant localizations. Cellular septins were resolved as localization pairs and thin stretches of equidistant localizations.

Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging.

Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color.

Single-molecule localization microscopy using mCherry.

We demonstrate the potential of the commonly used red fluorescent protein mCherry for single-molecule super-resolution imaging. mCherry can be driven into a light-induced dark state in the presence of a thiol from which it can recover spontaneously or by irradiation with near UV light. We show imaging of subcellular protein structures such as microtubules and the nuclear pore complex with a resolution below 40 nm. We were able to image the C-terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling. The photon yield for mCherry is comparable to that of the latest optical highlighter fluorescent proteins. Our findings show that the widely used mCherry red fluorescent protein and the vast number of existing mCherry fusion proteins are readily amenable to super-resolution imaging. This obviates the need for generating novel protein fusions that may compromise function or the need for external fluorescent labeling.

Tackling sample-related artifacts in membrane FCS using parallel SAF and UAF detection.

The complex shape and plasticity of cells is an intricate issue for the measurement of molecular diffusion in plasma membranes by fluorescence correlation spectroscopy (FCS). An important precondition for accurate diffusion measurements is a sufficient flatness of the membrane over the considered region and the absence of non-membrane-bound fluorescence diffusion. A method is presented to identify axial motion components caused by a non-ideal geometry of the membrane based on simultaneous measurement of the fluorescence emitted above and below the critical angle of the specimen/glass interface. Thereby, two detection volumes are generated that are laterally coincident, but differ in their axial penetration of the specimen. The similarity between the intensity tracks of the supercritical angle fluorescence (SAF) and the undercritical angle fluorescence (UAF) strongly depends on the membrane flatness and intracellular fluorescence, and can help to avoid sample-related artifacts in the diffusion measurement.

Supercritical angle fluorescence immunoassay platform.

An inexpensive and easy-to-use immunoassay platform for the sensitive detection of analytes is presented. It comprises single-use polymer test tubes and a compact fluorescence reader. The optics for the capture of supercritical angle fluorescence (SAF) has been built into the tubes allowing for the extremely sensitive readout of solid phase immunoassays in real time and without washing steps. One-step sandwich immunoassays with interleukin 2 (IL-2) were carried out with capture antibodies immobilized in the tubes. At a turn around time of 12 min, the limit of detection for IL-2 was 0.27 pM (4.5 pg/mL) and the linear range covered 3 orders of magnitude. The developed technology is also adaptable to well plates and has great potential of replacing the work-intensive and time-consuming enzyme-linked immunosorbant assay (ELISA).

Simultaneous near-field and far-field fluorescence microscopy of single molecules.

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy.

We report a noninvasive fluorescence microscopy method and demonstrate nanometer resolution along the optical axis. The technique is based on the influence of the microscope slide on the angular intensity distribution of fluorescence. Axial positions are determined by measuring the proportion of light emitted below the critical angle of total internal reflection, which behaves in a classical way, and light emitted above the critical angle, which is exponentially dependent on the distance of the fluorophore from the microscope slide.

Nanoscale Structural Plasticity of the Active Zone Matrix Modulates Presynaptic Function.

The active zone (AZ) matrix of presynaptic terminals coordinates the recruitment of voltage-gated calcium channels (VGCCs) and synaptic vesicles to orchestrate neurotransmitter release. However, the spatial organization of the AZ and how it controls vesicle fusion remain poorly understood. Here, we employ super-resolution microscopy and ratiometric imaging to visualize the AZ structure on the nanoscale, revealing segregation between the AZ matrix, VGCCs, and putative release sites. Long-term blockade of neuronal activity leads to reversible AZ matrix unclustering and presynaptic actin depolymerization, allowing for enrichment of AZ machinery. Conversely, patterned optogenetic stimulation of postsynaptic neurons retrogradely enhanced AZ clustering. In individual synapses, AZ clustering was inversely correlated with local VGCC recruitment and vesicle cycling. Acute actin depolymerization led to rapid (5 min) nanoscale AZ matrix unclustering. We propose a model whereby neuronal activity modulates presynaptic function in a homeostatic manner by altering the clustering state of the AZ matrix.

Activity-Dependent Gating of Parvalbumin Interneuron Function by the Perineuronal Net Protein Brevican.

Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.

Nanoscopic compartmentalization of membrane protein motion at the axon initial segment.

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal, and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axonal and somatodendritic domains. Recently, an axonal periodic pattern of actin, spectrin, and ankyrin forming 190-nm-spaced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear. Here, we performed single-particle tracking time-course experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single-particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton. We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ∼190-nm-spaced segments along the AIS axis as early as day in vitro 4, and this pattern alternates with actin rings. Mathematical modeling shows that diffusion barriers between the segments significantly reduce lateral diffusion along the axon.

Dual color single particle tracking via nanobodies.

Single particle tracking is a powerful tool to investigate the function of biological molecules by following their motion in space. However, the simultaneous tracking of two different species of molecules is still difficult to realize without compromising the length or density of trajectories, the localization accuracy or the simplicity of the assay. Here, we demonstrate a simple dual color single particle tracking assay using small, bright, high-affinity labeling via nanobodies of accessible targets with widely available instrumentation. We furthermore apply a ratiometric step-size analysis method to visualize differences in apparent membrane viscosity.

A simple method for GFP- and RFP-based dual color single-molecule localization microscopy.

The recent development of single-molecule localization-based super-resolution techniques has afforded a resolution in the nanometer range in light microscopy. The ability to resolve biological structures on this scale by multicolor techniques faces significant challenges which have prevented their widespread use. Here, we provide a generic approach for high-quality simultaneous two-color single-molecule localization microscopy imaging of any combination of GFP- and RFP-tagged proteins with the use of nanobodies. Our method addresses a number of common issues related to two-color experiments, including accuracy and density of labeling as well as chromatic aberration and color-crosstalk with only minimal technical requirements. We demonstrate two-color imaging of various nanoscopic structures and show a compound resolution down to the limit routinely achieved only in a single color.

Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence.

We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.