Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range, and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in its push-pull structure raises concerns about its potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ∼100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400 nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissues-lipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space, and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.