Covalent labeling of mitochondria with a photostable fluorescent thiol-reactive rhodamine-based probe

Fluorescent imaging of mitochondria is an essential tool for studies of mitochondrial functions. The staining of mitochondria with potential-indicating dyes, e.g., rhodamine 123, readily vanishes upon loss of the transmembrane potential under certain conditions. 1-(Rhodamine B)-4-(2′-chloroacetyl)-piperazine amide (RB-CAP) was shown to be electrophoretically accumulated into mitochondria, forming covalent bioconjugates with intramitochondrial protein sulfhydryls which enabled the mitochondrial staining to endure in subsequent collapse of the transmembrane potentials. RB-CAP is highly photostable and exhibits stringent selectivity in covalent labeling of mitochondria in living cells. Being much less expensive, RB-CAP is a superior substituent for MitoTracker probes in functional studies of mitochondria.