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 ¿M 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. Lay Description: BODIPY (4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene) is a widely used fluorescence molecule which can be used to label biological material such as proteins, lipids and nucleotides. Their optical properties are very attractive (for example they are very bright), however in spite of this they have a small separation between their maximum excitation and emission wavelengths (Stokes shift), which leads to a decrease in emission signal due to what is known as self quenching. Furthermore ideally they should emit in the Near Infra Red Region, where tissue transmits light well. Herein, we compare two BODIPY compounds from a recently developed family of naphthyridine substituted BODIPY derivatives, one with emission in the visible region of the spectra (BODIPY-VIS) and another with a near-infrared (NIR) emission. This NIR flourophore has a large Stokes shift of approximatly 165 nm in water, which overcomes the self quenching effect and can be used as a contrast reagent for live mammalian cells and murine brain tissue. The compounds were rendered water soluble by their bonding to polyethelene 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, localising predominantly within the lipophillic ("fat loving") organelles of the cells; the endoplasmic reticulum (ER) and mitochondria. Cytotoxicity studies revealed that these BODIPY derivatives induce modest cell death at concentrations exceeding 10 mM where each derivative induces a different mode of cell death, either by apoptotic or necrotic cell death pathways. Whereas the quantum yield (or brightness) of emission of the visible emitting fluorphore was over an order of magnitude greater than the large Stokes shifted NIR probe, the latter showed considerably reduced tendancy to self quench and less interference from autofluorescence. The NIR probe also showed good penetrability and staining in fixed and especially live tissue samples. In the latter case, a similar tendency to exclude the nucleus and to localise in the mitochondria and ER was observed as in live single cells. This to our knowledge is the first demonstration of such a larege Stokes shifted NIR BODIPY probe applied to cell and tissue imaging. © 2014 Royal Microscopical Society.