Eosin Y (Free Acid)

Technical Specifications

Product Description & Scientific Applications

Eosin Y (Free Acid) (2',4',5',7'-tetrabromofluorescein; C.I. 45380:2; Solvent Red 43) is the spirit-soluble free-acid form of the brominated xanthene dye eosin Y. Bromination at the 2',4',5',7' positions of the fluorescein scaffold gives the red-orange colour, shifts absorption toward green (λabs ≈ 520–540 nm, depending on form and medium), and introduces a heavy-atom effect that accelerates intersystem crossing to a long-lived triplet state. As the protonated free acid rather than the disodium salt, it dissolves preferentially in ethanol, methanol, and other polar organic media and has limited water solubility; aqueous eosin reagents instead use the water-soluble disodium salt. This makes the free acid the form for alcoholic and solvent-based eosin systems and for the organic-phase photochemistry below.

H&E and cytological staining (eosin Y chromophore). As an anionic dye under mildly acidic conditions, eosin Y is the acidic counterstain in haematoxylin and eosin (H&E) staining, binding positively charged, protein-rich components — cytoplasm, collagen, smooth and skeletal muscle, erythrocytes, keratin, and extracellular matrix — to give pink-to-red contrast against blue-purple haematoxylin-stained nuclei. The same chromophore is the eosin component of Papanicolaou EA and Romanowsky-type haematology stains, contributing erythrocyte colour, eosinophil-granule staining, and cytoplasmic contrast. These aqueous reagents typically use the disodium salt; the spirit-soluble free acid is the option where an alcoholic or solvent-based eosin is wanted, with differentiation during the water/alcohol rinse and dehydration and a little acid sharpening contrast.

Membrane-integrity viability (eosin Y chromophore). In dye-exclusion assays, live cells with intact plasma membranes exclude the anionic dye while cells with compromised membranes take it up and stain pink-to-red — complementary to trypan blue and erythrosine B. The eosin-nigrosin sperm vitality stain is included in WHO semen-analysis methodology, recommended in the 6th-edition framework when total sperm motility is below 40%, with nigrosin giving a dark bright-field background for scoring stained non-vital against unstained membrane-intact spermatozoa; the standard reagent uses water-soluble eosin.

Fluorescence imaging and pseudo-H&E. The four bromine substituents lower fluorescence quantum yield relative to fluorescein, but eosin Y remains a visible-emitting fluorophore (λem ≈ 540–550 nm) under green excitation, serving as the cytoplasmic/stromal channel in pseudo-H&E. With a nuclear fluorophore such as DRAQ5, it gives two-channel datasets that linear mixing and Beer–Lambert modelling convert into H&E-like images. The DRAQ5/eosin "D&E" workflow has been validated against conventional H&E on fixed and frozen sections and applied to fresh, unsectioned biopsy cores by optical-sectioning fluorescence microscopy, and related eosin-plus-nuclear-stain workflows run on structured illumination and open-top light-sheet microscopy.

Visible-light photoredox catalysis. Eosin Y is a widely used metal-free organic photocatalyst and an alternative to ruthenium and iridium polypyridyl complexes. Visible-light excitation populates the singlet state, then heavy-atom-enhanced intersystem crossing gives a long-lived triplet (lifetime ≈ 24 µs). For the anionic eosin Y manifold, excited-state potentials are about E°*(red) ≈ +0.8 V and E°*(ox) ≈ −1.1 V vs SCE, enabling both oxidative- and reductive-quenching (Type I single-electron-transfer) cycles. Because acid-base speciation changes with solvent, pH, base, and counterion, the free acid is best treated as an alcohol/organic-soluble precursor to the catalytically active eosin species under buffered, basic, or ion-paired conditions rather than as interchangeable with the disodium salt. It supports aryl-radical generation from diazonium salts, trifluoromethylation, sp³ C–H functionalisation of amines and ethers, oxidative heteroarene functionalisation, nitrogen-centred radical chemistry, ATRA-type additions to alkenes, aerobic cyclisations, and dehydrogenative cross-couplings. The same triplet drives Type II photosensitisation, transferring energy to ground-state O₂ to generate singlet oxygen for aerobic oxidations such as sulfide oxidation and for antimicrobial photodynamic inactivation. It is typically used at low mol% under inexpensive green-LED illumination.

Other uses. FRET acceptor and environment-sensitive fluorescent probe in supramolecular, surfactant, and dye-binding studies; photoinitiator/photosensitiser in visible-light radical photopolymerisation, including eosin/amine acrylate systems and eosin-mediated thiol-ene formulations.

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