BODIPY
Background
BOC Sciences is committed to providing customers with high-quality BODIPY dyes. BODIPY dyes are widely used in fluorescent probes, biomarkers and imaging, photosensitizers, solar cells, and light trapping.
What Does BODIPY Stain?
Boron-dipyrromethene (BODIPY) dyes are a class of near-infrared fluorescent dyes with excellent performance. Its molecular structure has a larger conjugated system, as well as greater planarity and rigidity. By modifying the molecular structure of BODIPY, a series of derivatives could be obtained for applications in life sciences, environmental monitoring and other fields.
BODIPY Structure
BODIPY dyes are a class of fluorescent dyes with a unique chemical structure. Their core structure consists of a boron-dipyrromethene moiety, which gives BODIPY dyes excellent optical properties. The molecular weight of BODIPY dyes typically ranges from 500 to 1000 Daltons, depending on the modification of their side chains. The symmetry and conjugated system of their molecular structure allow them to exhibit high brightness and low phototoxicity in fluorescence imaging.
Fig. 1. BODIPY structure and BODIPY spectrum (BOC Sciences Authorized).BODIPY Spectrum
The optical properties of BODIPY dyes form the basis for their wide application in biomedical research. The absorption and emission spectra of BODIPY dyes usually fall within the visible light range, enabling their detection under conventional fluorescence microscopes. For example, BODIPY 493/503 is a commonly used green fluorescent dye with a maximum absorption wavelength of 493 nm and a maximum emission wavelength of 503 nm. Due to its good water solubility and low cytotoxicity, this dye is widely used for cell imaging and lipid droplet staining. In addition, BODIPY dyes have high fluorescence intensity and excellent photostability, ensuring that their fluorescence signal does not significantly decay even under prolonged illumination.
Dye Name Excitation (nm) Emission (nm) Notes BODIPY 493/503 493 503 Commonly used for lipid droplet staining BODIPY 581/591 C11 581 (reduced) / 500–510 (oxidized) 591 (reduced) / 510–520 (oxidized) Emission shifts with oxidation state BODIPY 488 503 512 Similar to FITC, excitable by 488 nm laser BODIPY 493 493 503 Similar to BODIPY 493/503 BODIPY 500/510 500 510 Short-wavelength green fluorescent dye BODIPY 505/515 505 515 Common for neutral lipid staining (e.g., lipid droplets) BODIPY 558/568 558 568 Orange-red fluorescent dye BODIPY 576/589 576 589 Strong lipid-binding affinity BODIPY 581/591 581 591 Similar to C11, used to monitor oxidation BODIPY 630/650 630 650 Deep red fluorescence, suitable for multiplexing BODIPY 650/665 650 665 Near-infrared region, suitable for deep tissue imaging BODIPY 665/676 665 676 High tissue penetration, suitable for in vivo imaging BODIPY C11 ~581 ~591 Oxidation-sensitive, used for lipid peroxidation detection BODIPY C12 500–510 510–520 Used in lipid metabolism studies BODIPY C16 ~500–510 ~510–520 Long-chain fatty acid probe, used for tracking lipid metabolism Table 1. Absorption and emission spectral data of common BODIPY dyes. (Please note: Different suppliers may use slightly different chemical structures or labeling systems for dyes with the same name. Therefore, the exact absorption/emission peaks may vary by ±2–5 nm. It is recommended to consult the specific product datasheet before use.)
BODIPY Solubility
The physicochemical properties of BODIPY dyes significantly affect their applications. Solubility is one of the key factors influencing experimental design. Most BODIPY dyes are well soluble in organic solvents such as dimethyl sulfoxide (DMSO) and ethanol. However, through chemical modification, water-soluble BODIPY derivatives can also be synthesized, thus expanding their applicability in biomedical research. In addition, the molecular structure of BODIPY dyes is closely related to their fluorescence properties. For example, by altering the length of side chains and functional groups, the fluorescence wavelength and quantum yield can be tuned.
BODIPY Dye Synthesis and Modification
The synthesis of specific BODIPY derivatives according to experimental requirements is an important research direction. Researchers can design and synthesize BODIPY labels with specific affinities based on the properties of target biomolecules. This customized synthesis strategy not only improves experimental specificity but also provides new ideas for the development of novel biological probes.
BODIPY Synthesis Methods
The synthesis of BODIPY dyes typically involves constructing the core structure—the dipyrromethene scaffold—followed by coordination with boron trifluoride (BF₃·OEt₂) to form a stable BODIPY structure. Typical synthesis steps include:
- Synthesis of pyrrole derivatives: Functionalized pyrrole monomers are obtained through Fischer indole synthesis or other organic synthesis strategies.
- Formation of dipyrromethene: Two pyrrole monomers are condensed through aldehyde or ketone intermediates to form dipyrromethene.
- Oxidation and complexation: The intermediate is oxidized (e.g., using DDQ) to form dipyrromethene, followed by complexation with BF₃·OEt₂ to yield the typical fluorescent BODIPY core.
In addition, structural diversification can also be achieved by introducing side chains or functionalizing the scaffold sites. For example, introducing alkyl, aryl, or hydrophilic groups at the 3,5 or 8 positions of BODIPY can modulate its solubility, fluorescence properties, or targeting capabilities. Notably, Aza-BODIPY is an important class of BODIPY derivatives in which some carbon atoms are replaced by nitrogen atoms. This structural change significantly alters the spectral properties, especially enhancing absorption and emission in the near-infrared (NIR) region, making them valuable for deep-tissue imaging and photothermal therapy. Aza-BODIPY also shows high photostability and environmental sensitivity, making it suitable for constructing multifunctional responsive fluorescent probes.
BODIPY Derivatives Development
Developing structurally diverse and functionally rich BODIPY derivatives is a key direction to promote their wide applications. By introducing various functional or bioactive groups onto the BODIPY scaffold, a series of specialized molecules can be obtained for applications in biomarking, drug delivery, sensing, and energy materials. Examples include:
- BODIPY-Cholesterol: Incorporation of a cholesterol group allows the derivative to insert selectively into the lipid bilayer of cell membranes via hydrophobic interactions, enabling selective membrane labeling widely used in membrane dynamics studies and visualization.
- BODIPY Amine: A BODIPY molecule containing a primary amine (–NH₂) group, providing a reactive site for chemical modification and covalent conjugation with proteins or small molecules, expanding its use in fluorescent probes and biological labeling.
- BODIPY Azide: A BODIPY derivative with an azide (–N₃) group that participates in highly efficient and specific click chemistry reactions (e.g., CuAAC), widely used for biomolecule labeling, material modification, and complex molecule construction.
- BODIPY Carboxylic Acid: A BODIPY molecule bearing a carboxyl (–COOH) group that enables covalent linkage via amide bonds to amine-containing molecules like proteins and antibodies, commonly applied in bioimaging and functional probe design.
- BODIPY Ceramide: A derivative where BODIPY is attached to ceramide, a key lipid in cell membranes and signaling, allowing visualization of lipid distribution and metabolic dynamics within cells.
- BODIPY Cyclopamine: A BODIPY-labeled derivative of cyclopamine, a Hedgehog pathway inhibitor, combining pharmacological activity and fluorescence for cellular studies of distribution and targeting.
- BODIPY Fatty Acid: BODIPY conjugated with fatty acid chains of various lengths, exhibiting lipophilicity that facilitates insertion into membranes or lipid droplets for studies of lipid metabolism, uptake, transport, and imaging.
- BODIPY Maleimide: A BODIPY derivative containing a maleimide group that reacts specifically with thiol (–SH) groups such as cysteine residues, commonly used for labeling proteins, peptides, and sulfur-containing biomolecules.
- BODIPY NHS Ester: A BODIPY derivative with an active N-hydroxysuccinimide (NHS) ester group that reacts with primary amines to form stable amide bonds, widely used for fluorescent labeling of proteins and other amine-containing molecules in biochemical research.
BODIPY Staining Principle
The staining principle of BODIPY dyes is mainly based on their unique chemical structure and excellent optical properties. These dyes exhibit high lipophilicity, enabling them to selectively bind to lipid-rich structures such as cell membranes and lipid droplets, thereby achieving targeted staining of cellular substructures. Meanwhile, BODIPY dyes have high fluorescence quantum yield, strong photostability, and narrow emission spectra, producing clear and bright imaging under fluorescence microscopy. Through specific chemical modifications, BODIPY dyes can also achieve covalent or non-covalent binding with proteins, nucleic acids, and other biomolecules, allowing more precise biological labeling.
BODIPY Staining Protocol
BODIPY staining is a commonly used fluorescence method for labeling intracellular lipids, membrane structures, or specific biomolecules. It features high sensitivity, low background, and excellent photostability. The method relies on the lipophilicity and outstanding fluorescence properties of BODIPY dyes to provide clear and stable signals in cell imaging. BODIPY staining is widely applied in lipid metabolism studies, cell structure analysis, and live-cell imaging. It is suitable for various sample types such as fixed cells, tissue sections, or live cells.
BODIPY Staining Fixed Cells
BODIPY staining of fixed cells is a common experimental method. The procedure typically involves cell fixation, staining, and microscopic observation. Formaldehyde is a commonly used fixative to preserve cellular structures and prevent deformation during staining. The staining process usually involves incubating fixed cells with BODIPY dye at a specific concentration for a set duration. For example, when staining lipid droplets with BODIPY 493/503, cells are typically incubated in dye solution for 30 minutes. Dye concentration and incubation time should be optimized to avoid over- or under-staining.
BODIPY Staining Live Cells
Live-cell staining is another important application of BODIPY dyes. Unlike fixed cell staining, live-cell staining must be performed while maintaining cell viability. The low phototoxicity and good cell compatibility of BODIPY dyes make them ideal for live-cell imaging. During live-cell staining, the dye typically enters cells via passive diffusion and binds to intracellular lipid structures. For example, in lipid droplet staining, BODIPY dyes selectively bind to the lipid bilayer of droplets, clearly revealing their distribution under the microscope. Experimental design for live-cell staining should consider cell type, dye concentration, and incubation time to ensure reliable and reproducible results.
BODIPY Staining Tissue
BODIPY dyes are also widely used in tissue section staining. Preparation of tissue sections involves fixation, dehydration, and embedding. During staining, BODIPY dyes bind to lipid structures in the tissue and visualize lipid distribution under the microscope. For example, in adipose tissue sections, BODIPY dyes specifically bind to lipid droplets, clearly revealing adipocyte structures. BODIPY dyes can also be combined with other staining methods, such as hematoxylin and eosin (H&E) staining, to provide comprehensive tissue structural information.
BODIPY Dye Advantages
Compared with rhodamine or cyanine dyes, BODIPY dyes offer several superior properties:
- High molar extinction coefficient and strong light sensitivity;
- High fluorescence quantum yield (usually >0.6) with low self-quenching;
- Stable spectral properties, minimally affected by pH and solvent polarity;
- Narrow emission peak, high detection sensitivity;
- Excellent photostability, thermal, and chemical stability;
- Low molecular weight and cytotoxicity;
- Easily modifiable molecular structure;
- Long excitation wavelength, typically in the near-infrared region.
BODIPY Dye Applications
BODIPY-based ion and molecular probes can detect various inorganic or organic ions and molecules with high efficiency and sensitivity. BODIPY dyes are also widely used as fluorescent markers in protein detection, nucleic acid hybridization sequencing, and disease diagnostics. Additionally, functionalized BODIPY dyes can generate singlet oxygen, showing potential as photosensitizers in photodynamic therapy. Beyond biochemical labeling, BODIPY dyes are also used in lasers, dye-sensitized solar cells, and light-trapping systems.
BODIPY Lipid Droplet
Lipid droplets are key organelles for intracellular lipid storage. BODIPY dyes have a unique advantage in staining lipid droplets by selectively binding to the lipid bilayer. Common dyes include BODIPY 493/503 and BODIPY 550/565. These dyes not only reveal lipid droplet distribution but also indicate droplet size and number via fluorescence intensity changes. For example, during lipid metabolism research, BODIPY dyes can be used to observe droplet formation, fusion, and degradation.
BODIPY Lipid Probes
BODIPY dyes can serve as lipid probes in lipid metabolism research. By chemical modification, BODIPY lipid probes with specific affinity can be synthesized. These probes bind selectively to lipid molecules and can be detected under microscopy. For instance, during lipid transport studies, BODIPY lipid probes track intracellular lipid dynamics. They are also useful for investigating lipid-protein interactions, revealing molecular mechanisms of lipid metabolism.
BODIPY Live Cell Imaging
BODIPY dyes are widely used in cell imaging due to low phototoxicity and good biocompatibility, making them ideal for live-cell applications. In flow cytometry, BODIPY dyes assist in cell sorting and cell cycle analysis. For instance, by labeling cell membranes with BODIPY dyes, rapid cell sorting can be achieved. BODIPY dyes also enable visualization of dynamic cellular processes, such as in cell migration assays where cell membranes are labeled to track movement.
Experimental Case Studies of BODIPY Dyes
BODIPY Lipid Droplet Staining Protocol
Materials and Reagents
- Cell line: 3T3-L1 adipocytes;
- BODIPY dye: BODIPY 493/503;
- Other reagents: DMEM medium, insulin, dexamethasone, triglycerides.
Staining Steps and Results
- Cell culture: Culture 3T3-L1 cells in DMEM to 80% confluency;
- Differentiation induction: Add insulin, dexamethasone, and triglycerides to induce adipocyte differentiation;
- Staining: Wash differentiated cells with PBS and incubate with 1 μM BODIPY 493/503 for 30 minutes;
- Microscopy: Observe lipid droplet distribution under a fluorescence microscope. Results show clear visualization of droplet size and number with stable signal.
BODIPY Live Cell Imaging Protocol
Experimental Design and Cell Culture
- Cell line: HeLa cells;
- BODIPY dye: BODIPY-cholesterol;
- Other reagents: DMEM medium, fetal bovine serum.
Dye Labeling and Fluorescence Imaging
- Cell culture: Grow HeLa cells to 70% confluency in DMEM;
- Dye labeling: Wash cells with PBS, then incubate with 2 μM BODIPY-cholesterol for 15 minutes;
- Microscopy: Observe membrane fluorescence using live-cell imaging. Results show specific labeling of the cell membrane and clear visualization of its structure.
BODIPY Staining Tissue Protocol
Tissue Section Preparation
- Tissue: Mouse adipose tissue;
- Fixative: 4% formaldehyde;
- Embedding medium: Paraffin.
BODIPY Staining and Microscopy
- Fixation and embedding: Fix tissue in 4% formaldehyde for 24 hours, then embed in paraffin;
- Sectioning and staining: Deparaffinize 5 μm tissue sections and incubate with 1 μM BODIPY 493/503 for 30 minutes;
- Microscopy: Use a fluorescence microscope to observe lipid droplet distribution. Results show clear structures of lipid droplets in adipocytes.
Conclusion
With unique optical properties and broad biocompatibility, BODIPY dyes play a significant role in biomedical research. From lipid droplet staining to live-cell imaging, tissue section staining to multimodal imaging, their application scope continues to expand. With ongoing development of novel BODIPY derivatives and multimodal imaging techniques, BODIPY dyes will have even greater potential in biomedical applications. In the future, BODIPY dyes are expected to play a vital role in clinical diagnosis and therapy, contributing to human health.
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