The Brilliant Violet™ family of fluorescent molecules are organic polymers with an extraordinary capacity to absorb energy (extinction coefficient) and a high efficiency with which to convert that absorbed energy to an emitted signal (quantum yield). When conjugated to antibodies, this results in high intensity brightness on labeled cells.

Improvements over Traditional Organic Dyes
Traditional organic dyes are quite small, ranging between 300 - 1200 Da. Although multiple dyes can be conjugated to a single antibody, each acts independently from the others around them, thus their potency is limited to their own structural constraints and the potential for self quenching. In contrast, the Brilliant Violet™ polymers are comparable in size to AmCyan or APC, but unlike a protein, consist of repeating fluorescent subunits that act cooperatively along the entire length of the polymer backbone. Energy is conducted much like a molecular antennae to capture light and pass it down the antennae like a lightning rod. As the subunits work cooperatively, the polymers' extinction coefficient is substantially larger than that of standard organic dyes and this is reflected in superior brightness and signal-to-noise.

Brilliant Violet™ Chemistry
Structurally, Brilliant Violet™ polymers are unsaturated organic materials comprised of alternating single and double bonds and aromatic units. It is this repeating bond structure that creates a continuous π-orbital system and extended electronic delocalization. Such features give rise to unique and tunable optical properties, including large extinction coefficients (>10⁶ M^-1cm^-1), intense photoluminescence, and massive collective response, all of which help to address fundamental limitations in detection sensitivity. Adaptation of polymer side chain chemistry to impart solubility in aqueous solution confers ability for use as a highly sensitive fluorescent conjugate in biological applications such as flow cytometry and microscopy.

Physical properties, such as high quantum yield in typical flow buffers, high solubility, and minimal non-specfic binding, are all built into the backbone structure and in the side chain modifications. Additionally, the polymer design specifically incorporates well defined functional sites for covalent attachment to antibodies.

High Sensitivity Fluorescence™
Flourochromes derived from conducting polymers represent a new paradigm, High Sensitivity Fluorescence™, which aims to address fundamental shortcomings in reagents currently available for violet excitation in flow cytometry. Common dyes like Pacific Blue™, Alexa Fluor® 488 and Cy5 have high quantum yields but limited extinction coefficients. Alternative reporters like phycobiliproteins offer much greater absorbance cross-sections, producing brighter signals, but are limited by rapid photobleaching and sensitivity to fixation. Because the extinction coefficient of a conjugated polymer is directly proportional to the degree of polymerization (or number of repeat units), these structures are designed for improved brightness. Further, as these materials are derived from common synthetic organic and polymer chemistry techniques it is possible to manufacture reagents which are more defined and reproducible, in terms of size, conjugation sites, physical properties, and optical properties.

Unlike quantum dots, conjugated polymers have discrete excitation spectra, similar to that of organic dyes, which minimizes potential issues with cross-beam compensation.

Physical Properties
Brilliant Violet 421™ has an extinction coefficient of 2,500,000 M^-1cm^-1 at 405 nm, an aqueous solution quantum yield of 65 ± 5% and solubility in excess of 50 mg/mL in PBS. The extinction coefficient contributes to its superior brightness compared to Pacific Blue™, which has an extinction coefficient of 30,000 M^-1cm^-1.

High Sensitivity Fluorescence™ polymers can also be modified with functional groups to produce high stokes shift emissions. Brilliant Violet 570™, Brilliant Violet 605™, and Brilliant Violet 650™ are such variants of the Brilliant Violet 421™ polymer, emitting maximally at 570 nm, 603 nm, and 645 nm, respectively. The figure below provides the excitation and emission spectra of the Brilliant Violet™ fluorophores. These can also be compared to other fluorophores in our Spectra Analyzer tool.

Brilliant Violet™ antibody conjugates will change the future of flow cytometry, bringing new power to the violet laser. In particular, Brilliant Violet 421™ antibodies consistently stain at brighter levels than PE, the brightest known fluorochrome, bringing unparalleled sensitivity and resolution to the violet laser, while Brilliant Violet 570™ antibodies add needed versatility to panel selection for multicolor flow cytometry. Brilliant Violet 605™ and Brilliant Violet 650™ antibodies also provide exceptionally bright signal and further expand options for multicolor panels. For a guide on how to use Brilliant Violet™ conjugates in flow cytometry, download our technical sheet: Brilliant Violet™ Considerations for Multicolor Flow Cytometry.

Easy to Use and Trouble-Free
Brilliant Violet™ antibodies are simple to use, compatible with standard staining buffers, and stable to fixation. Provided in convenient 5 µl test sizes at optimal ready-to-use concentrations, our Brilliant Violet™ antibody products can easily be added to your multi-color panels. To compare fluorescence spectra data of Brilliant Violet™ conjugates with our other fluorochromes use the BioLegend Fluorescence Spectra Analyzer.

Where BD Horizon™ V450, Pacific Blue™ and eFluor® 405 are just too dim for fluorescence microscopy, Brilliant Violet 421™ now enables visualization of antigens with directly conjugated antibodies, enhancing your capabilities with multi-color microscopy.