A nucleolus-targeting hill-type pH probe

Abstract Nucleolus is a sub-cellular nuclear structure identified with an optical microscope. Surprisingly, the fluorescent labelling tools for nucleolus remains very scarce compared to other cellular organelles. In recent years, the biorelevance of nucleolus in various diseases receive intensity scrutiny, and therefore high-performance fluorescence stains for nucleolus are warranted. The proximal pH of the cell nucleolus is slightly lower compared to the other nucleoplasmic region. However, differentiation of a minor pH variation with a traditional Henderson-Hasselbalch type pH probe is difficult due to its inferior sensitivity. Harnessing a molecular mechanism for pH-sensing with positive cooperativity, we were able to design Hill-type small-molecular pH probes with a profile. They exhibit a reduced acid-base transition range and better differentiate biorelevant small pH changes. We showcase that such Hill-type pH probes are suitable for selective nucleolus staining. It exhibits fast membrane permeability and applies to live, fixed, and permeated cells. PHN is expected to find applications in nucleolar chemical biological studies.