BODIPY 493/503

Technical Specifications

Product Description & Scientific Applications

BODIPY 493/503 (Pyrromethene 546; PM 546; PMPBF2) is 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene — a boron difluoride complex of a pentamethyl-substituted dipyrromethene carrying no ionisable substituent. That neutrality is the working property: the dye is nonpolar and electrically uncharged, which is why it partitions into neutral lipid rather than into charged membranes.

Absorption and emission maxima are quoted as 493 and 503 nm, and they move with medium: 493 and 506 nm in acetone with a fluorescence quantum yield of 0.84, against 500 and 512 nm in cyclohexane with a quantum yield of 0.91. Laser-grade material is used as a quantum-yield reference standard at 0.85 in ethanol. Emission is narrow and largely confined to the band passed by standard green filters, and the Stokes shift is only 10 nm.

Lipid droplet imaging. The most commonly used lipid droplet tracker, established in fixed cells and equally usable live, effective in fixed and unfixed preparations and unaffected by permeabilisation. Fluorescence is very specific for lipid droplets with one documented exception: in lean cells the dye may accumulate in cellular membranes, so droplet-poor samples need their own controls. Substantial overlap with LysoTracker has also been reported in HepG2 cells, indicating non-specific lysosomal staining. Added at 1 µl of a 1 mg/ml stock per ml of secondary antibody solution, it allows the droplet core and a specific intracellular protein to be visualised in one sample.

Flow cytometry and quantitation. Mean fluorescence intensity gives a quantitative readout of droplet content, validated against PLIN2 knockdown. In bacterial polyhydroxybutyrate measurement it does not saturate above 25 pg per cell, where Nile Red does — the case where this dye is the better quantitative choice.

Choosing between lipid stains. Nile Red generally fluoresces more intensely and photobleaches less rapidly; LD540 has been developed with higher specificity and photostability than either. Against LipidTOX the comparison runs the other way: this dye gives superior intensity, while LipidTOX bleaches rapidly and does not resolve droplet morphology.

Photobleaching and spectral overlap. Bleaching is fast under continuous excitation — roughly 70% signal loss in 20 seconds and 90% after 240 seconds in HepG2 cells under laser illumination. The 10 nm Stokes shift and the 493/503 band also mean the dye cannot be spectrally separated from ECFP, EGFP or EYFP, which rules out multicolour live-cell imaging alongside those proteins.

Laser and photophysical use. Sold laser-grade at greater than 99% purity, and used as Pyrromethene 546 in dye-laser work at millimolar concentration. Under transversal pumping at 355 nm, lasing efficiencies of 23% in ethyl acetate, 18% in ethanol and 14% in acetone have been reported. It has also been doped into PDMS host matrices for solid-state laser applications.

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