The Application of Water Soluble, Mega-Stokes-shifted BODIPY Fluorophores to Cell and Tissue Imaging

BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores are widely used in bioimaging to label proteins, lipids and nucleotides, but in spite of their attractive optical properties they tend to be prone to self-quenching because of their notably small Stokes shift. Herein, we compare two BODIPY compounds from a recently developed family of naphthyridine substituted BODIPY derivatives, one a visible emitting derivative (BODIPY-VIS) and one a near-infrared emitting fluorophore with a Stokes shift of approximately 165 nm as contrast reagents for live mammalian cells and murine brain tissue. The compounds were rendered water soluble by their conjugation to polyethylene glycol (PEG). Both PEGylated compounds exhibited good cell uptake compared with their parent compounds and confocal fluorescence microscopy revealed all dyes explored to be nuclear excluding, localizing predominantly within the lipophilic organelles; the endoplasmic reticulum and mitochondria. Cytotoxicity studies revealed that these BODIPY derivatives are modestly cytotoxic at concentrations exceeding 10 μM where they induce apoptosis and necrosis. Although the quantum yield of emission of the visible emitting fluorophore was over an order of magnitude greater than the Mega-Stokes shifted probe, the latter showed considerably reduced tendency to self quench and less interference from autofluorescence. The near-infrared probe also showed good penetrability and staining in live tissue samples. In the latter case similar tendency to exclude the nucleus and to localize in the mitochondria and endoplasmic reticulum was observed as in live cells. This to our knowledge is the first demonstration of such a Mega-Stokes BODIPY probe applied to cell and tissue imaging.