Resonance Raman imaging of the NADPH oxidase subunit cytochrome b ₅₅₈ in single neutrophilic granulocytes

We have employed confocal resonance Raman (RR) imaging to visualize the subcellular distribution of the NADPH oxidase subunit cytochrome b₅₅₈ in both resting and phagocytosing neutrophils. Our Raman microscopic technique is a label-free, chemical (vibrational) imaging method that can be applied to individual, intact cells, thus probing cytochrome b₅₅₈ in its native environment. The Raman signal from cytochrome b₅₅₈ is resonantly and selectively enhanced in neutrophils by using 413 nm excitation. Experiments on resting neutrophils show a cytoplasmic distribution of cytochrome b₅₅₈, with several areas of high content. Upon phagocytosis of polystyrene particles, we found that part of the cytochrome b₅₅₈ is translocated toward the ingested beads. This is in accordance with immunocytochemistry studies combined with electron and fluorescence microscopy. As compared to these methods, RR microscopy requires minimal sample preparation and disturbance. Moreover, it allows the determination of the redox state of cytochrome b₅₅₈ inside the cell, which reflects its NADPH oxidase activity.