Overview of the BODIPY fluorophores

The BODIPY fluorophores have spectral characteristics that are often superior to those of fluorescein, tetramethylrhodamine, Texas Red and longer-wavelength dyes. With derivatives that span the visible spectrum (Figure 1.4.1), BODIPY dyes are extremely versatile.ref We use them to generate fluorescent conjugates of proteins, nucleotides, oligonucleotides and dextrans, as well as to prepare fluorescent enzyme substrates, fatty acids, phospholipids, lipopolysaccharides, receptor ligands and polystyrene microspheres.

BODIPY dyes are unusual in that they are relatively nonpolar and the chromophore is electrically neutral (Figure 1.4.2). These properties tend to minimize dye-induced perturbation of conjugate functional properties. BODIPY dyes are therefore often the preferred choice for labeling nucleotides, amino acids and other low molecular weight ligands.ref BODIPY dye conjugates of low molecular weight molecules also tend to be more permeant to live cells than are conjugates of charged fluorophores (Membrane-Permeant Reactive Tracers—Section 14.2). With their high peak intensity, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis.ref BODIPY dyes are also more useful than most other long-wavelength dyes, including fluoresceins and carbocyanines, for assays that measure fluorescence polarization ref (Fluorescence Polarization (FP)—Note 1.4), and they have large cross-sections for excitation by multiphoton excitation sources ref (Fluorescent Probes for Two-Photon Microscopy—Note 1.5).


Figure 1.4.1 Normalized fluorescence emission spectra of 1) BODIPY FL, 2) BODIPY R6G, 3) BODIPY TMR, 4) BODIPY 581/591, 5) BODIPY TR, 6) BODIPY 630/650 and 7) BODIPY 650/665 fluorophores in methanol.


Figure 1.4.2 The structure and numbering of the BODIPY fluorophore, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.

The core structure of the BODIPY fluorophore is shown in Figure 1.4.2. Solutions of the alkyl-substituted derivatives have a green, fluorescein-like fluorescence. However, when substituents that yield additional conjugation are added to the parent molecule, both the absorption and emission spectra of the resulting derivatives can shift to significantly longer wavelengths, with emission maxima of greater than 750 nm now possible with some BODIPY derivatives. Our goal has been to develop BODIPY dyes that are optimal for the major excitation sources and that match the common optical filter sets. Accordingly, our recommended BODIPY substitutes for the fluorescein, rhodamine 6G, tetramethylrhodamine and Texas Red fluorophores are named BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, respectively (Figure 1.4.3). Because there are so many different BODIPY dyes, we have had to develop a systematic strategy for naming them. Except for BODIPY FL, BODIPY R6G, BODIPY TMR and BODIPY TR, we identify these dyes with the registered trademark BODIPY followed by the approximate absorption/emission maxima in nm (determined in methanol); for example, the BODIPY 581/591 dye.

Amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7) are discussed below; thiol-reactive BODIPY dyes are included in Thiol-Reactive Probes Excited with Visible Light—Section 2.2. Other reactive BODIPY dyes useful for derivatizing aldehydes, ketones and carboxylic acids are described in Reagents for Modifying Aldehydes and Ketones—Section 3.3 and Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4. Applications of some thiol-reactive BODIPY dyes for cell tracing are discussed in Membrane-Permeant Reactive Tracers—Section 14.2.

BODIPY FL dye: a substitute for fluorescein

With the most fluorescein-like spectra of the BODIPY dyes, the green-fluorescent BODIPY FL fluorophore (excitation/emission maxima ~503/512 nm) has several characteristics ref that make it potentially superior to fluorescein in some applications. These include:

  • High extinction coefficient (EC >80,000 cm-1M-1) and high fluorescence quantum yield (often approaching 1.0, even in water)
  • Lack of ionic charge and spectra that are relatively insensitive to solvent polarity and pH ref
  • Narrow emission bandwidth (Figure 1.4.3), resulting in a higher peak intensity than that of fluorescein
  • Red shift in fluorescence emission at high dye concentrations—a property that can be used to detect regions of high probe density ref (photo)
  • Relatively long excited-state lifetime (typically 5 nanoseconds or longer), which is useful for fluorescence polarization–based assays (Fluorescence Polarization (FP)—Note 1.4)
  • Little or no spectral overlap with longer-wavelength dyes such as tetramethylrhodamine and Texas Red dye (Figure 1.4.3), making BODIPY FL one of the preferred green-fluorescent dyes for multicolor applications ref
  • Greater photostability than fluorescein in some environments ref
  • Large two-photon cross-section for multiphoton excitation ref (Fluorescent Probes for Two-Photon Microscopy—Note 1.5)

Figure 1.4.3 Normalized fluorescence emission spectra of goat anti–mouse IgG antibody conjugates of fluorescein (FL), tetramethylrhodamine (TMR) and the Texas Red (TR) dyes, shown by dashed lines (---), as compared with goat anti–mouse IgG antibody conjugates of BODIPY FL, BODIPY TMR and BODIPY TR dyes, respectively, shown by solid lines (—).

Longer-wavelength BODIPY dyes

It is possible to synthesize BODIPY fluorophores with altered spectral properties by simply changing the substituents on the parent molecule. This discovery has led to creation of a series of longer-wavelength BODIPY dyes with fluorescence spectra that span the visible spectrum (Figure 1.4.1). The BODIPY R6G (excitation/emission maxima ~528/547 nm), BODIPY TMR (excitation/emission maxima ~543/569 nm) and BODIPY TR (excitation/emission maxima ~592/618 nm) fluorophores are spectrally similar to the rhodamine 6G, tetramethylrhodamine and Texas Red fluorophores, respectively, and are thus compatible with standard optical filter sets designed for these important dyes.

The BODIPY 630/650-X and BODIPY 650/665-X fluorophores are the longest-wavelength amine-reactive BODIPY fluorophores currently available. The spectral properties of these longer-wavelength BODIPY derivatives retain most of the advantages of the BODIPY FL fluorophore, including narrow bandwidths, high extinction coefficients, good fluorescence quantum yields and relatively long excited-state lifetimes (>3 nanoseconds for the BODIPY 630/650 dye ref). Like the BODIPY FL fluorophore, however, most of these dyes have a small Stokes shift, which may require that they be excited or detected at suboptimal wavelengths. The spectral characteristics of 13 different red-fluorescent fluorophores, including the Alexa Fluor 647 (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3) and BODIPY 630/650 dyes, have been evaluated in different surrounding media to assess the influence of polarity, viscosity and detergent concentration and to facilitate probe choice in fluorescence-based assays.ref

Amine-reactive BODIPY dyes

BODIPY succinimidyl esters

We offer an extensive selection of amine-reactive BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7). These include succinimidyl esters of several BODIPY propionic acids and of BODIPY FL pentanoic acid:

  • BODIPY FL C3 succinimidyl ester (D2184)
  • BODIPY FL C5 succinimidyl ester (D6184)
  • BODIPY 493/503 C3 succinimidyl ester (D2191)
  • BODIPY 530/550 C3 succinimidyl ester (D2187)
  • BODIPY 558/568 C3 succinimidyl ester (D2219)
  • BODIPY 576/589 C3 succinimidyl ester (D2225)
  • BODIPY 581/591 C3 succinimidyl ester (D2228)

We have also prepared reactive BODIPY X succinimidyl esters that contain an additional seven-atom aminohexanoyl spacer ("X") between the fluorophore and the succinimidyl ester group. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents such as anti-dye antibodies.ref These BODIPY X succinimidyl esters include:

  • BODIPY FL-X succinimidyl ester (D6102)
  • BODIPY TMR-X succinimidyl ester (D6117)
  • BODIPY TR-X succinimidyl ester (D6116)
  • BODIPY 630/650-X succinimidyl ester (D10000)
  • BODIPY 650/665-X succinimidyl ester (D10001)

BODIPY succinimidyl esters are particularly useful for preparing conjugates of peptides, nucleotides, oligonucleotides, drugs, toxins, sphingolipids and other low molecular weight ligands that contain aliphatic amines.ref Several BODIPY succinimidyl esters have been conjugated to aminoacyl tRNAs for metabolic incorporation into proteins through in vitro translation.ref

BODIPY TMR-X SE has been reacted with a series of peptide ligands for use in a high-throughput fluorescence polarization assay of ligand binding to G protein–coupled receptors.ref The red fluorescence of the BODIPY 581/591 fluorophore shifts to green fluorescence upon peroxidation, a unique feature that has been exploited for ratiometric measurements of lipid oxidation in live cells (Generating and Detecting Reactive Oxygen Species—Section 18.2). BODIPY 630/650-X and BODIPY 650/665-X succinimidyl esters (D10000, D10001) are quite fluorescent when conjugated to nucleotides ref and oligonucleotides and can be excited with near-infrared excitation sources.

For amplifying the BODIPY FL dye signal or converting it into an electron-dense signal, we offer an unlabeled anti–BODIPY FL rabbit polyclonal antibody (A5770, Anti-Dye and Anti-Hapten Antibodies—Section 7.4). This antibody crossreacts with some other BODIPY dyes, but not with other fluorophores, and therefore should not be used for simultaneous detection of more than one dye based on the BODIPY fluorophore.

Water-soluble BODIPY sulfonated succinimidyl esters

The moderate lipophilicity of the BODIPY propionic acid succinimidyl esters discussed above requires their dissolution in an organic solvent before use in conjugations. Although these reactive dyes are very useful for preparing conjugates of amines in organic solvents, they are less suitable for reaction with proteins.

To address the solubility in aqueous solution, we have prepared the sulfosuccinimidyl ester of BODIPY FL propionic acid (BODIPY FL SSE, D6140), as well as the STP ester of BODIPY FL propionic acid (B10006). STP esters,ref which are prepared by coupling a carboxylic acid and 4-sulfo-2,3,5,6-tetrafluorophenol (Figure 1.4.4), are more readily purified than sulfosuccinimidyl esters but equally amine reactive. BODIPY FL SSE and BODIPY FL STP ester are quite soluble in water and more suitable than the corresponding BODIPY succinimidyl esters for amine conjugation in aqueous solution. These sulfonated succinimidyl esters are useful for preparing conjugates of proteins, amine-modified oligonucleotides and other biomolecules.ref

Figure 1.4.4 Reaction of a primary amine with an STP ester.

BODIPY carboxylic acids

Two green-fluorescent BODIPY carboxylic acids (D2183D3834) are available. These carboxylic acid derivatives can be converted to fluorescent esters,ref acid halides or amides using standard chemical techniques.

BODIPY dye conjugates and their applications

The versatility of the BODIPY fluorophore is demonstrated by its incorporation into literally hundreds of products listed in this Handbook, including many of our FluoSpheres and TransFluoSpheres microspheres (Microspheres—Section 6.5), enzyme substrates (Enzyme Substrates and Assays—Chapter 10) and several of our imaging and flow cytometry standards (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1, Flow Cytometry Reference Standards—Section 23.2). Some examples of our BODIPY dye conjugates are described below.

BODIPY peptide, protein and polysaccharide conjugates

As is common with many fluorescent dyes, conjugation of BODIPY dyes to proteins is sometimes accompanied by significant fluorescence quenching.ref Because of this potential problem, we do not recommend using the simple BODIPY propionic acid succinimidyl esters discussed above for preparing most protein conjugates, although peptides labeled with a single BODIPY dye can be quite fluorescent and are quite useful for fluorescence polarization–based assays ref (Labeling Small Peptides with Amine Reactive Dyes in Organic Solvents—Note 9.2). We prepare conjugates of its BODIPY dyes with an exceptionally wide variety of peptides, proteins and polysaccharides, including:

In addition, we prepare conjugates of proteins (and of starch) that are so heavily labeled that they are almost nonfluorescent (Figure 1.4.7). Use of the EnzChek Kits and DQ reagents that incorporate these bioconjugates as fluorogenic enzyme substrates is described later in this section and in Detecting Peptidases and Proteases—Section 10.4.

BODIPY nucleotide and oligonucleotide conjugates

With the exception of guanosine nucleotides (see below), fluorescence quenching is usually not a problem if the BODIPY derivative is conjugated to nucleotides, oligonucleotides, peptides or low molecular weight amines in which the stoichiometry of modification is 1:1. BODIPY FL dye–labeled oligonucleotide primers also have lower photodestruction rates than fluorescein-labeled primers, improving the detectability of labeled DNA in sequencing gels.ref Oligonucleotide conjugates of several of our BODIPY dyes have been shown to be useful for DNA sequencing ref (Labeling Oligonucleotides and Nucleic Acids—Section 8.2, Molecular Probes nucleic acid labeling kits—Table 8.6), in part because the dye exhibits minimal effect on the mobility of the fragment during electrophoresis.ref

In addition, we have prepared BODIPY FL conjugates of ATP and GTP that are labeled through the ribose moieties and serve as structural probes of nucleotide-binding proteins (A12410, G12411; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3). The fluorescence of BODIPY dyes is quenched by photoinduced electron transfer from proximal guanosine bases.ref BODIPY FL GTP therefore shows significant fluorescence quenching (Figure 1.4.5) that is relieved by binding to GTP-binding proteins (G-proteins).

For protein-binding studies that require nonhydrolyzable nucleotides, we offer the BODIPY FL fluorophore linked through the γ-thiol of ATP-γ-S (A22184) and the γ-thiol of GTP-γ-Sref (G22183, Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3). Like BODIPY FL GTP, the fluorescence of the BODIPY GTP-γ-S thioesters is quenched ~90% relative to that of the free dye but is recovered upon protein binding to at least some G-proteins.ref The green-fluorescent BODIPY FL GTP-γ-S has been used to detect GTP-binding proteins separated by capillary electrophoresis.ref BODIPY 515/530 GTP-γ-S thioester also exhibits green fluorescence and has a greater fluorescence increase upon protein binding, as compared with the BODIPY FL GTP-γ-S thioester. The BODIPY TR GTP-γ-S thioester is a red-fluorescent analog with spectral properties similar to those of the Texas Red dye.

In addition to their potential use for binding studies, the BODIPY FL ATP-γ-S and BODIPY FL GTP-γ-S thioesters are important substrates for Fhit (Figure 1.4.6), a member of the histidine triad superfamily of nucleotide-binding proteins that bind and cleave diadenosine polyphosphates.ref Fhit, one of the most frequently inactivated proteins in lung cancer, functions as a tumor suppressor by inducing apoptosis.ref These BODIPY nucleotides should be especially useful for screening potential Fhit inhibitors and activators.


Figure 1.4.5 Fluorescence emission spectra of (1) free BODIPY FL dye in phosphate-buffered saline, pH 7.2; (2) BODIPY FL ATP (A12410); and (3) BODIPY FL GTP (G12411). Samples were prepared with equal absorbance at the excitation wavelength (488 nm). The areas under the curves are therefore proportional to the relative fluorescence quantum yields, clearly showing the quenching effect caused by interaction of the BODIPY FL fluorophore with the guanine base of GTP.

Figure 1.4.6 Principle of fluorescence-based detection of the diadenosine triphosphate hydrolase activity of Fhit using BODIPY FL GTP-γ-S thioester (G22183) as a substrate analog.

BODIPY lipids and BODIPY receptor ligand conjugates

BODIPY dye conjugates of lipids, toxins, steroids, drug analogs and receptor probes typically have quantum yields approaching unity, especially in organic solvents. The low polarity of the BODIPY fluorophore makes probes containing these dyes excellent analogs of biological lipids (Probes for Lipids and Membranes—Chapter 13). Consequently, these probes are well tolerated by enzymes that metabolize lipids, including phospholipases and sphingomyelinases (Probes for Lipid Metabolism and Signaling—Section 17.4Fluorescence-based phospholipase assays—Table 17.3). In most cases, lack of a spectral shift in the metabolic product's fluorescence requires use of an easy extraction and chromatographic separation step to detect product formation, with quantitation possible by photography or with a fluorescence- or absorption-based scanner. BODIPY derivatives of lipids and low molecular weight ligands include analogs of:

In addition to the BODIPY dye conjugates of receptor ligands in the list above, we have utilized BODIPY dyes for synthesis of several LysoTracker and LysoSensor dyes, which are extremely useful probes for labeling acidic organelles in live cells. These products are discussed in Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3.

DQ reagents: heavily labeled BODIPY dye conjugates as fluorogenic enzyme substrates

We have found BODIPY dye conjugates to be very useful reagents for numerous bioanalytical screening applications. In particular, we have utilized the tendency of BODIPY dyes to quench their fluorescence on conjugation to certain biopolymers to our advantage (Figure 1.4.7) in the following enzyme-assay kits and reagents:

Conjugation of either the BODIPY FL dye (excitation/emission maxima ~500/506 nm) or BODIPY TR dye (excitation/emission maxima ~589/617 nm) to a biopolymer at high degrees of substitution (DOS) results in almost total quenching of the conjugate's fluorescence; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Enzyme-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent BODIPY FL dye– or BODIPY TR-X dye–labeled peptides (Figure 1.4.7), or, in the case of the BODIPY FL amylase substrate in the EnzChek Ultra Amylase Assay Kit, BODIPY FL dye–labeled carbohydrates. The increase in fluorescence, which can be measured with a spectrofluorometer, minifluorometer or fluorescence microplate reader, is proportional to enzymatic activity. The DQ BSA and DQ ovalbumin substrates are particularly suitable for the study of receptor labeling and antigen processing. DQ BSA conjugates can be targeted to Fc receptors after they are complexed with our anti-BSA antibody (A11133Anti–Epitope Tag and Anti-Reporter Antibodies—Section 7.5). Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing. Upon endocytosis and proteolysis, highly fluorescent peptides are released within intracellular vacuoles. DQ ovalbumin appears to be an excellent indicator of macrophage-mediated antigen processing in flow cytometry and microscopy assays.


Figure 1.4.7 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek Protease Assay Kits relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.

BODIPY dye conjugates for fluorescence polarization–based assays

When a fluorescent molecule tethered to a protein is excited by polarized fluorescent light, the polarization of fluorescence emission is dependent on the rate of molecular tumbling. Upon proteolytic cleavage of the fluorescently labeled protein, the smaller peptides that result tumble faster and the emitted light is depolarized relative to the light measured from the intact conjugate (Fluorescence Polarization (FP)—Note 1.4). Fluorescence polarization technology is more sensitive than many other nonradioactive assays for proteases and allows measurements to be taken in real time, permitting the collection of kinetic data.

The relatively long fluorescence lifetimes (typically >5 nanoseconds) at visible wavelengths, good anisotropy properties, high molar absorptivity and fluorescence intensity and lack of pH sensitivity in the spectra of the BODIPY dyes have been shown to make these dyes the preferred fluorophores for high-throughput fluorescence polarization–based assays. For example, we have developed a green-fluorescent BODIPY FL casein substrate with an optimal degree of labeling for fluorescence polarization–based protease assays (contact Custom Services for more information). BODIPY dye conjugates of nucleotides, peptides and drug analogs are available or are readily prepared from the chemically reactive BODIPY dyes. Fluorescence polarization–based assays for G-protein–coupled receptors, kinases and phosphatases and for high-affinity receptors are particularly important when screening for new drug candidates.

Additional methods of analysis using BODIPY dye conjugates

In addition to their general utility for the intensity-based and fluorescence polarization–based assays described above, the BODIPY dyes are near optimal for a variety of other bioanalytical techniques:

  • The spectral variety and high absorbance of the BODIPY dyes (Figure 1.4.1) permits their use as efficient donor or acceptor dyes for numerous assays that use fluorescence resonance energy transfer, including internally quenched endopeptidase substrates ref (Detecting Peptidases and Proteases—Section 10.4), nucleic acid hybridization assays and receptor-binding assays (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).
  • BODIPY dye conjugates of peptides are readily separated by chromatographic means and can be used to detect the activity of enzymes that catalyze secondary modifications, such as phosphorylation/dephosphorylation, glycosylation/deglycosylation, oxidation/reduction, myristoylation, farnesylation and peptide–peptide crosslinking.
  • Hydrolysis of peptides that are singly labeled with BODIPY dyes to smaller peptides can be detected chromatographically with extremely high sensitivity.
  • With their high peak intensity and narrow emission spectra, reactive BODIPY dyes are among the most detectable amine-derivatization reagents available for HPLC and capillary electrophoresis; thus, amine-containing metabolites can be derivatized with succinimidyl esters of the BODIPY dyes (Amine-reactive BODIPY dyes—Table 1.7) for ultrasensitive analysis.ref

Data Table

For a detailed explanation of column headings, see Definitions of Data Table Contents

Cat #MWStorageSolubleAbsECEmSolventNotes
542.19F,D,LH2O, DMSO50280,000510MeOH1, 2
292.09F,LDMSO, MeCN50591,000511MeOH1
389.16F,D,LDMSO, MeCN50282,000510MeOH1, 3
BODIPY 530/550 C3 SE
513.31F,D,LDMSO, MeCN53477,000551MeOH1
BODIPY 493/503 C3 SE
417.22F,D,LDMSO, MeCN50079,000509MeOH1
BODIPY 558/568 C3 SE
443.23F,D,LDMSO, MeCN55997,000568MeOH1
BODIPY 564/570 C3 SE463.25F,D,LDMSO, MeCN563142,000569MeOH1
BODIPY 576/589 C3 SE
426.19F,D,LDMSO, MeCN57583,000588MeOH1
BODIPY 581/591 C3 SE
489.28F,D,LDMSO, MeCN581136,000591MeOH4
320.15F,LDMSO, MeCN50596,000511MeOH1
502.32F,D,LDMSO, MeCN50485,000510MeOH1
634.46F,D,LDMSO, MeCN58868,000616MeOH1, 5
608.45F,D,LDMSO, MeCN54460,000570MeOH1
491.20F,D,LH2O, DMSO50275,000510MeOH1, 6
BODIPY R6G SE437.21F,D,LDMSO, MeCN52870,000547MeOH1
417.22F,D,LDMSO, MeCN50487,000511MeOH1
BODIPY 630/650-X SE
660.50F,D,LDMSO, MeCN625101,000640MeOH1, 7
BODIPY 650/665-X SE
643.45F,D,LDMSO, MeCN646102,000660MeOH1
  1. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  2. This sulfotetrafluorophenyl (STP) ester derivative is water soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due to hydrolysis.
  3. The fluorescence lifetime (τ) of D2184 in MeOH at 20°C is 5.7 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  4. Oxidation of the polyunsaturated butadienyl portion of the BODIPY 581/591 dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm.ref
  5. The fluorescence lifetime (τ) of D6116 in MeOH at 20°C is 5.4 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  6. This sulfonated succinimidyl ester derivative is water soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due to hydrolysis.
  7. The fluorescence lifetime (τ) of the BODIPY 630/650 dye at 20°C is 3.9 nanoseconds in H2O and 4.4 nanoseconds in EtOH.ref

For Research Use Only. Not for use in diagnostic procedures.