BODIPY

BODIPY refers to a class of fluorophores known for their high fluorescence quantum yield and strong molar extinction coefficients, enhancing their stability during experiments (Zhang & Zhu, 2019). Notably, BODIPY dyes are less sensitive to light compared to other fluorescent dyes; the unsubstituted BODIPY dye has a fluorescence lifetime of 7.2 nanoseconds (Schmitt et al., 2008). Because of these properties, they have become a widely used method for visualizing and quantifying lipid content in biological samples (Courtis et al., 2014).

The use of BODIPY dyes extends beyond lipid visualization. They serve multiple roles in biological research, acting as metal ion probes with a strong affinity for alkali metal ions such as magnesium (Mg²⁺) and sodium (Na⁺). Additionally, BODIPY derivatives have been developed into molecular probes for detecting nitric oxide (NO), a crucial signaling molecule in many physiological processes (Zhang et al., 2013). One of the prominent features of BODIPY dyes is their selectivity for glutathione (GSH), making them valuable for assessing GSH levels in living cells. The modifiable structure of these dyes helps create derivatives such as BODIPY 488. Furthermore, BODIPY dyes can act as molecular switches; for example, phenyl selenium-substituted BODIPY fluorescent switches (BOD-phse) have been shown to effectively detect hydrogen sulfide (H₂S) in cellular environments. In addition to these uses, BODIPY can be utilized in conjunction with delivery systems, such as Lipofectamine, which is chemically known as 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA).

When compared to other fluorescent dyes, BODIPY dyes offer several key advantages that make them highly effective as biological markers, including low photodegradation, charge neutrality, and minimal toxicity. Moreover, the hydrophobic characteristics of BODIPYs allow them to effectively penetrate cell membranes (Kand et al., 2020). However, despite these advantages, BODIPY dyes also have limitations. They exhibit high-energy absorption and emission, as well as relatively short Stokes shifts of approximately 5-15 nm. These characteristics can lead to light scattering, potentially resulting in a lower signal-to-noise ratio during imaging. (Yang et al., 2016)

In terms of biomolecule detection, BODIPY dyes excel at identifying a range of targets, including cholesterol, hydrogen nitroxide species (HNO), amyloid-β aggregates, and reactive nitrogen species (RNS; Nguyen et al., 2021). Various detection methods utilize fluorescent probes specific to HNO and RNS, along with assays like the BODIPY cholesterol assay (Bernecic et al., 2019). Their hydrophobic nature enables BODIPY dyes to bind effectively to different targets, including lipids and cellular membranes. Additionally, certain BODIPY conjugates can interact with drugs, proteins, and hormones, further enhancing their application uses.

BODIPY dyes, such as BODIPY FL and BODIPY 493/503, are common examples of BODIPY derivatives. BODIPY FL is characterized by its green fluorescence and excitation/emission maxima around 503/512 nm (Qin et al., 2004). It possesses a high extinction coefficient (EC > 80,000 cm⁻¹M⁻¹) and can achieve a quantum yield of 0.9 (Holicek, 2020). In contrast, BODIPY 493/503 serves as a tracer for neutral lipids, emitting a green fluorescence signal upon binding.

If one chooses to carry out BODIPY staining, the first step in the procedure involves dissolving the dye in an organic solvent, such as DMSO, DMF, or methanol. For instance, to prepare a 2 μM BODIPY staining solution, a 5 mM stock solution can be created by dissolving 1.3 mg of BODIPY in 1 ml of DMSO, which is stable for storage at -20 °C (Qiu & Simon, 2016). Following preparation, cells should be washed with 3 ml of PBS to remove any media or serum. The BODIPY staining solution is then incubated with the cells for 15 to 30 minutes at room temperature, after which the cells are rinsed briefly with PBS to remove excess dye. Finally, the samples are mounted on a microscope coverslip for visualization using a fluorescence microscope.

BODIPY

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