Antibodies Conjugated with GlyCLICK for Super Resolution Imaging

Super resolution microscopy techniques such as stimulated emission depletion microscopy (STED) improves imaging resolution compared to conventional light microscopy. In STED microscopy, super resolution is achieved using photoactivatable dyes that are excited and de-excited selectively with a laser to restrict fluorescense to a specific focal point. While such super resolution methods in combination with immunostaining advances the quality of imaging, limitations related to dyes and conjugation strategies remain.


In this paper, Weber and colleagues at University Göttingen synthesized a new fluorophore suitable for super resolution microscopy techniques. The ONB-2SiR dye is photoactivatable while being stable against two-photon activation, routinely used in STED microscopy. The scientists also present an efficient conjugation and purification protocol for labeling antibodies with hydrophobic and aggregating dyes using the GlyCLICK technology and phase separation.


To obtain a defined degree of labeling (DOL) and compensate the hydrophobic nature of the fluorophore, a conjugation and purification protocol suitable for aggregating and poorly soluable dyes was developed. Using the GlyCLICK technology, antibodies were labeled specifically at the Fc glycan sites, generating conjugates with preserved affinity carrying two labels per antibody. Compared to other conjugation methods, GlyCLICK resulted in conjugates with a definite DOL of 2 while also reducing aggregation. The conjugates were then purified using a tree-phase separation technique, allowing the scientists to purify the hydrophobic dye from the labeled antibodies.


In summary, the scientists present a new fluorophore suitable not only for STED, but for other single-molecule localization microscopy techniques such as PALM and MINIFLUX. This work also highlights the importance of high-quality conjugation, which is obtained using GlyCLICK.


Weber et al., 2020. Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels. Chemistry – A European Journal. doi: 10.1002/chem202004645


Site-specific labeling of native IgG using a single kit.