A new class of bioorthogonal labeling probes was developed based on Bicyclo[6.1.0]non-4-yne (BCN), a strained cycloalkyne that displays excellent reaction kinetics in cycloadditions with azides and nitrones. Functionalization with Alexa Fluor 555 or biotin facilitated straightforward and highly specific visualization of proteins and glycans, and allowed subcellular-resolved imaging of glycan expression in metastatic melanoma cells during invasive migration into 3D collagen lattices.