Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.

Area of Science:

• Glycobiology
• Cellular Imaging
• Bioorthogonal Chemistry

Background:

• Glycosylation is crucial for molecular recognition and implicated in various diseases.
• Metabolic oligosaccharide engineering (MOE) is vital for studying glycans, both intracellularly and on cell surfaces.
• High-resolution imaging of intracellular glycans is essential but often limited by microscopy accessibility.

Purpose of the Study:

• To demonstrate expansion microscopy for visualizing intracellular sialylated glycans.
• To combine MOE with click chemistry for bioorthogonal labeling of glycans.
• To provide a protocol for enhanced intracellular glycan imaging.

Main Methods:

• Metabolic oligosaccharide engineering (MOE) for incorporating bioorthogonal reporters into glycans.
• Click chemistry for targeted bioconjugation of molecular dyes.
• Expansion microscopy for physical enlargement of labeled biological specimens.

Main Results:

• Successful visualization of intracellular sialylated glycans using MOE and expansion microscopy.
• Demonstrated feasibility of bioorthogonal labeling targeting sialylation.
• Enabled co-localization studies with immunofluorescence.

Conclusions:

• Expansion microscopy offers an accessible method for high-resolution imaging of intracellular glycans.
• The proposed protocol facilitates studies on sialoconjugate biosynthesis, trafficking, and recycling.
• This approach overcomes limitations of traditional super-resolution microscopy for glycan analysis.