Look beyond - Detect your proteins with SYSY antibodies directly coupled to abberior STAR dyes

What could be better for your super-resolved imaging research than top quality primary antibodies, validated by renowned researchers from all over the world, combined with high-quality dyes, designed to perfectly meet the specific requirements of the STED super-resolution microscope. Here we present a panel of the most widely used SYSY primary antibodies in the field of neuroscience, directly coupled with a matching pair of abberior STAR dyes, optimized for STED microscopy.

SYSY antibodies that meet the specific requirements of the STED super-resolution microscopy

Overview

• Introduction
• Neurotransmission/ The structure of a chemical synapse
• STED microscopy
• Prerequisites for successful STED imaging
• Advantages of direct coupling
• Products
• Literature

Introduction

Unravelling structure and function of synapses has been long conducted successfully. Nonetheless, the diffraction barrier in conventional microscopy techniques limited resolution and thus insights in structure and function of a synapse. Super-resolution microscopy enables unprecedented precision in visualizing synaptic structures. This capability transforms our ability to examine the spatial organization of proteins within the synaptic cleft, active zones, and postsynaptic densities in great detail. It facilitates the study of the heterogeneity and dynamics of synaptic components at the molecular level.

Neurotransmission/ The structure of a chemical synapse

Neurons must transmit information rapidly and accurate; they are responsible for quickly conveying signals from every part of our body, from the tips of our toes to the brain, where this information is processed. To achieve such fast and precise communication, synapses optimize their capacity to handle high demands and intermittent signal processing. 

A chemical synapse, the main type of synapse found in vertebrates, is a discontinuous structure, consisting of a presynaptic neuron and a postsynaptic neuron, separated by a synaptic cleft approximately 15–20 nm wide. Signal transmission at chemical synapses occurs through the synaptic vesicle cycle. Specialized proteins found in synaptic vesicles and at the active zone play crucial roles in coordinating the synaptic vesicle cycle. SNARE proteins, such as Syntaxin, Synaptobrevin, and SNAP-25, are involved in the fusion of synaptic vesicles with the plasma membrane. Scaffold proteins like Bassoon, Piccolo, and RIM organize and recruit synaptic vesicles to the vicinity of the plasma membrane. Synapsins aid in the movement of synaptic vesicles by binding to cytoskeletal proteins, while complexins prevent the spontaneous fusion of vesicles. Additionally, CAPS (Calcium-Activated Protein for Secretion) facilitate the coupling of calcium signaling to the fusion of synaptic vesicles (Chua et al., 2010, Jahn and Fasshauer, 2012).

The postsynaptic density (PSD) is a specialized region abundant in proteins, pivotal for receiving neurotransmitter signals from the presynaptic neuron and initiating postsynaptic responses. Enriched within the PSD are neurotransmitter receptors, comprising a diverse population at each synapse, predominantly determined by the neurotransmitter released by the presynaptic neuron. This receptor-released neurotransmitter pairing governs the electrical properties of each specific synapse. Additionally, various classes of scaffold proteins, such as PSD-95, Shank, and Homer, play crucial roles in anchoring and organizing signalling molecules, receptors, and structural proteins within the postsynaptic density (Takamori et al., 2006, Chua et al., 2010).

The synaptic cleft is primarily filled with extracellular matrix and other non-proteinaceous components. Among them are the cell-adhesion molecules, like cadherins, integrins, neuroligins (found in the postsynaptic membrane) and neurexins (found in the presynaptic membrane). Together, they mediate synaptic adhesion and play roles in synapse formation and function. Additional extracellular matrix proteins like laminins, collagens, and proteoglycans help organizing and stabilize synaptic connections (Dankovich and Rizzoli, 2022).

Advancements in microscopy technologies, particularly super-resolution techniques, have made it possible to visualize synapses in remarkable detail. These technologies have allowed scientists to observe specific protein interactions and the heterogeneity of synapses and synaptic vesicles, significantly deepening our understanding of these complex biological structures (Binotti et al., 2024, Upmanyu et al., 2022, Nishimune et al., 2016, Willig et al., 2006). However, many aspects of synaptic structure and function remain unresolved. For instance, the fine architecture of the presynaptic active zone (AZ), the significance of molecular clusters and the dynamics and molecular mechanisms underlying synaptic vesicle cycle are still incompletely described (Nosov et al., 2020).

STED microscopy

Before the advent of nanoscopic light microscopy techniques, particularly the development of STED (Stimulated Emission Depletion) microscopy, researchers were constrained by the diffraction limit of traditional light microscopy. This limit restricted resolution to approximately 200-300 nanometer laterally and about 500-700 nanometer axially. The concept of STED microscopy was proposed by Stefan Hell and Jan Wichmann in 1994 and was experimentally realized in 2000 (Hell and Wichmann, 1994, Klar et al., 2000). STED microscopy is based on a conventional confocal laser scanning configuration but includes, in addition to an excitation laser, a second red-shifted and donut-shaped STED laser with zero intensity in its center. This way, all molecules except the ones in the center of the donut are depleted. This configuration ensures that fluorophores excited by the central beam are rapidly de-excited by the STED beam while any fluorescence comes from fluorophores in the center of the STED donut. The fluorescence signal is sharpened and a lateral resolution of 30-70 nanometer is reached. The diffraction barrier is broken.

Prerequisites for successful STED imaging

A good imaging result depends on two conditions: good fluorescent dyes and specific antibodies. An ideal fluorescent probe for STED must possess several key attributes: responsiveness to the STED beam for efficient de-excitation, optimized absorption and emission spectra that align with available laser lines in STED systems, and resistance to photobleaching during repeated excitation and de-excitation cycles. For optimal STED imaging, abberior, the pioneer company in STED dyes, offers specially optimized fluorescent dyes. Renowned for their exceptional photostability, tailored spectral properties, efficient depletion capabilities, high quantum yield, compatibility with biological samples, and minimal background noise, abberior STAR ORANGE and STAR RED are the perfect duo for two-color STED @775 nm.
In achieving high spatial resolution in STED microscopy, the choice of antibodies used to stain live or fixed samples is crucial. SYSY antibodies show high specificity and affinity, making them precise and powerful tools for your microscopy analyses. Using well-characterized monoclonal antibodies, binding to a single epitope with high specificity, reduces variability in staining patterns and the potential for cross-reactivity with other proteins or molecules.
 

Advantages of direct coupling

In contrast to indirect labeling using labeled secondary antibodies, direct coupling displays a series of major benefits. Next to eliminating the need for a secondary antibody, direct coupling reduces steric hindrance, which in indirect labeling can decrease the primary antibody's affinity and specificity to the target. Moreover, it decreases background noise through unspecific secondary binding to non-target proteins (cross-reactivity), reduces the distance between fluorophore and antigen, and prevents masking of spatial arrangements of individual molecules by multiple secondaries binding to a single primary antibody (Früh et al., 2021).

Elevate your STED microscopy experience further with primary antibodies from SYSY conjugated to abberior STAR dyes.

SYSY antibodies that meet the specific requirements of the STED super-resolution microscopy

Literature

Binotti et al., 2024: ATG9 resides on a unique population of small vesicles in presynaptic nerve terminals. PMID: 37881948

Chua et al., 2010: The architecture of an excitatory synapse. PMID: 20200227

Dankovich and Rizzoli, 2022: The Synaptic Extracellular Matrix: Long-Lived, Stable, and Still Remarkably Dynamic. PMID: 35350469

Früh et al., 2021: Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors. PMID: 34184536

Hell and Wichmann, 1994: Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. PMID: 19844443

Jahn and Fasshauer 2012: Molecular machines governing exocytosis of synaptic vesicles. PMID: 23060190

Klar et al., 2000: Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. PMID: 10899992

Nishimune et al., 2016: Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice. PMID: 27321892

Nosov et al., 2020: The Decade of Super-Resolution Microscopy of the Presynapse. PMID: 32848695

Takamori et al., 2006: Molecular anatomy of a trafficking organelle. PMID: 17110340

Upmanyu et al., 2022: Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3. PMID: 35263617

Willig et al., 2006: STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. PMID: 16612384