Amphiphilic Rhodamine and Fluorescein Derivatives

Each of our amphiphilic probes comprises a moderately polar fluorescent dye with a lipophilic "tail." When used to stain membranes, including liposomes, the lipophilic portion of the probe tends to insert in the membrane and the polar fluorophore resides on the surface (Figure 13.2.1 in Fatty Acid Analogs and Phospholipids—Section 13.2), where it senses the membrane's surface environment and the surrounding medium.ref Our lipophilic carbocyanines and styryl dyes (Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4) are also amphiphilic molecules with a similar binding mode.

This section includes the classic membrane probes DPH, TMA-DPH, ANS, bis-ANS, TNS, prodan, laurdan and nile red, and also some lipophilic BODIPY and Dapoxyl dyes developed in our laboratories. Although they bear little resemblance to natural products, these probes tend to localize within cell membranes or liposomes or at their aqueous interfaces, where they are often used to report on characteristics of their local environment, such as viscosity, polarity and lipid order.

Octadecyl Rhodamine B

The relief of the fluorescence self-quenching of octadecyl rhodamine B (O246, structure) can be used to monitor membrane fusion ref—one of several experimental approaches developed for this application (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). Octadecyl rhodamine B has been reported to undergo a potential-dependent "flip-flop" from one monolayer of a fluid-state phospholipid bilayer membrane to the other, with partial relief of its fluorescence quenching.ref Investigators have used octadecyl rhodamine B in conjunction with video microscopy ref or digital imaging techniques ref to monitor viral fusion processes. Membrane fusion can also be followed by monitoring fluorescence resonance energy transfer to octadecyl rhodamine B from an acylaminofluorescein donor such as 5-hexadecanoylaminofluorescein ref (H110).

Fluorescence resonance energy transfer from fluorescein or dansyl labels to octadecyl rhodamine B has been used for structural studies of the blood coagulation factor IXa, EGF receptor and receptor-bound IgE.ref Octadecyl rhodamine B has also been used to stain kinesin-generated membrane tubules,ref to characterize detergent micelles,ref to assay for lysosomal degradation of lipoproteins ref and to investigate the influence of proteins on lipid dynamics using time-resolved fluorescence anisotropy.ref

Amphiphilic Fluoresceins

The amphiphilic fluorescein probes bind to membranes with the fluorophore at the aqueous interface and the alkyl tail protruding into the lipid interior. 5-Dodecanoylaminofluorescein (D109) is the hydrolysis product of our ImaGene Green C12-FDG β-galactosidase substrate (D2893, Detecting Glycosidases—Section 10.2). We also offer the homologous membrane probe 5-hexadecanoylaminofluorescein ref (H110, structure) and the octadecyl ester of fluorescein ref (F3857, structure).

Amphiphilic fluorescein probes are commonly used for fluorescence recovery after photobleaching (FRAP) measurements of lipid lateral diffusion.ref Some researchers have reported that 5-hexadecanoylaminofluorescein stays predominantly in the outer membrane leaflet of epithelia and does not pass through tight junctions, whereas the dodecanoyl derivative can "flip-flop" to the inner leaflet at 20°C (but not at <10°C) and may also pass through tight junctions.ref More recent studies have indicated that the lack of tight junction penetration of 5-hexadecanoylaminofluorescein is due to probe aggregation rather than a significant difference in its transport properties.ref

DPH and DPH Derivatives

Diphenylhexatriene (DPH)

1,6-Diphenyl-1,3,5-hexatriene (DPH, D202; structure) continues to be a popular fluorescent probe of membrane interiors. We also offer the cationic DPH derivative TMA-DPH (see below), as well as the phospholipid analog (D476, Fatty Acid Analogs and Phospholipids—Section 13.2). The orientation of DPH within lipid bilayers is loosely constrained. It is generally assumed to be oriented parallel to the lipid acyl chain axis (Figure 13.2.1A in Fatty Acid Analogs and Phospholipids—Section 13.2), but it can also reside in the center of the lipid bilayer parallel to the surface, as demonstrated by time-resolved fluorescence anisotropy and polarized fluorescence measurements of oriented samples.ref DPH shows no partition preference between coexisting gel- and fluid-phase phospholipids.ref Intercalation of DPH and its derivatives into membranes is accompanied by strong enhancement of their fluorescence; their fluorescence is practically negligible in water. The fluorescence decay of DPH in lipid bilayers is complex.ref Fluorescence decay data are often analyzed in terms of continuous lifetime distributions,ref which are in turn interpreted as being indicative of lipid environment heterogeneity.

DPH and its derivatives are cylindrically shaped molecules with absorption and fluorescence emission transition dipoles aligned approximately parallel to their long molecular axis. Consequently, their fluorescence polarization is high in the absence of rotational motion and is very sensitive to reorientation of the long axis resulting from interactions with surrounding lipids. These properties have led to their extensive use for membrane fluidity measurements.ref The exact physical interpretation of these measurements has some contentious aspects. For instance, the probes are largely sensitive to only the angular reorientation of lipid acyl chains—a motion that does not necessarily correlate with other dynamic processes such as lateral diffusion.ref Reviews on this subject ref should be consulted for further discussion. Time-resolved fluorescence polarization measurements of lipid order are more physically rigorous because they allow the angular range of acyl chain reorientation ("lipid order") to be resolved from its rate, and considerable research has been devoted to the interpretation of these measurements.ref


Designed to improve the localization of DPH in the membrane, TMA-DPH (T204, structure) contains a cationic trimethylammonium substituent that acts as a surface anchor.ref Like DPH, this derivative readily partitions from aqueous dispersions into membranes and other lipid assemblies, accompanied by strong fluorescence enhancement. The lipid–water partition coefficient (Kp) for TMA-DPH (Kp = 2.4 × 105) is lower than for DPH (Kp = 1.3 × 106), reflecting the increased water solubility caused by the polar substituents.ref The fluorescence decay lifetime of TMA-DPH is more sensitive to changes in lipid composition and temperature than is the fluorescence decay lifetime of DPH.ref

Staining of cell membranes by TMA-DPH is much more rapid than staining by DPH; however, the duration of plasma membrane surface staining by TMA-DPH before internalization into the cytoplasm is quite prolonged.ref As a consequence, TMA-DPH introduced into Madin–Darby canine kidney (MDCK) cell plasma membranes does not diffuse through tight junctions and remains in the apical domain, whereas the anionic DPH propionic acid accumulates rapidly in intracellular membranes.ref TMA-DPH residing in the plasma membrane can be extracted by washing with medium, thus providing a method for isolating internalized probe and monitoring endocytosis ref (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). Furthermore, because TMA-DPH is virtually nonfluorescent in water and binds in proportion to the available membrane surface,ref its fluorescence intensity is sensitive to increases in plasma membrane surface area resulting from exocytosis.ref

TMA-DPH fluorescence polarization measurements can be combined with video microscopy to provide spatially resolved images of phospholipid order in large liposomes and single cells.ref Information regarding lipid order heterogeneity among cell populations can be obtained in a similar way using flow cytometry.ref

Nonpolar BODIPY Probes

BODIPY Fluorophores

BODIPY fluorophore derivatives offer an unusual combination of nonpolar structure (structure) and long-wavelength absorption and fluorescence.ref BODIPY dyes have small fluorescence Stokes shifts, extinction coefficients that are typically greater than 80,000 cm-1M-1 and high fluorescence quantum yields that are not diminished in water.ref These dyes have applications as stains for neutral lipids and as tracers for oils and other nonpolar liquids. In addition, their photostability is generally high; this, together with other favorable characteristics (very low triplet–triplet absorption), make the BODIPY 493/503 and BODIPY 505/515 fluorophores excellent choices for flashlamp-pumped laser dyes.ref

Staining with the BODIPY 493/503 dye (D3922, structure) has been shown by flow cytometry to be more specific for cellular lipid droplets than staining with nile red ref (N1142). The low molecular weight of the BODIPY 493/503 dye (262 daltons) results in the probe having a relatively fast diffusion rate in membranes.ref The BODIPY 493/503 dye has also been used to detect neutral compounds in a microchip channel separation device.ref

BODIPY 505/515 (D3921, structure) rapidly permeates cell membranes of live zebrafish embryos,ref selectively staining cytoplasmic yolk platelets. This staining provides dramatic contrast enhancement of cytoplasm relative to nucleoplasm and interstitial spaces, allowing individual cell boundaries and cell nuclei to be imaged clearly with a confocal laser-scanning microscope (photo).

The very long–wavelength BODIPY 665/676 dye (B3932, structure) has fluorescence that is not visible to the human eye; however, it has found use as a probe for reactive oxygen species ref (Generating and Detecting Reactive Oxygen Species—Section 18.2).

BODIPY FL C5-Ceramide

BODIPY FL C5-ceramide (D3521, B22650; Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3) stains the plasma membrane, Golgi apparatus and cytoplasmic particles within the superficial enveloping layer (EVL) of the embryos. Once the fluorescent lipid percolates through the EVL epithelium, however, it remains localized within the interstitial fluid of the embryo and freely diffuses between cells (photo). Vital staining with BODIPY FL C5-ceramide thus allows hundreds of cells to be imaged en masse during morphogenetic movements.ref

CellTrace BODIPY TR Methyl Ester

Many research and biotechnological applications require detailed three- and four-dimensional visualization of embryonic cells labeled with green-fluorescent protein (GFP) within their native tissue environments. Fluorescent counterstains that label all the cells in a living embryo provide a histological context for the GFP-expressing cells in the specimen. The red-fluorescent CellTrace BODIPY TR methyl ester (C34556, structure) is an excellent counterstain for cells and tissues that are expressing GFP.ref This dye readily permeates cell membranes and selectively stains mitochondria and endomembranous organelles such as endoplasmic reticulum and the Golgi apparatus, but does not appear to localize in the plasma membrane. These localization properties make the dye an ideal vital stain that can be used to reveal: (1) the location and shapes of cell nuclei, (2) the shapes of cells within embryonic tissues and (3) the boundaries of organ-forming tissues within the whole embryo.ref Furthermore, CellTrace BODIPY TR methyl ester staining is retained after formaldehyde fixation and permeabilization with Triton X-100, and the dye does not appear to produce any teratogenic effects on embryonic development. The emission spectra of enhanced GFP (EGFP) and CellTrace BODIPY TR methyl ester are well separated, with peaks at 508 nm and 625 nm, respectively (Figure 13.5.1), allowing simultaneous dual-channel confocal imaging without significant overspill of GFP fluorescence into the CellTrace BODIPY TR methyl ester detection channel.

The Image-iT LIVE Intracellular Membrane and Nuclear Labeling Kit (I34407, Tracers for Membrane Labeling—Section 14.4) provides the red-fluorescent CellTrace BODIPY TR methyl ester along with the blue-fluorescent Hoechst 33342 dye for highly selective staining of the intracellular membranes and nuclei, respectively, of live or fixed cells or tissues (photo). These two fluorescent stains were especially chosen for their compatibility with live GFP-expressing cells, and they can be combined into one staining solution to save labeling time and wash steps while still providing optimal staining.



Figure 13.5.1 Normalized absorption (—) and fluorescence emission (---) spectra of enhanced green-fluorescent protein (EGFP) and CellTrace BODIPY TR methyl ester (C34556).

Pyrene, Nile Red and Bimane Probes

Nonpolar Pyrene Probe

1,3-Bis-(1-pyrene)propane (B311, structure) has two pyrene moieties linked by a three-carbon alkylene spacer. This probe is somewhat analogous to the bis-pyrenyl phospholipids (Fatty Acid Analogs and Phospholipids—Section 13.2) in that excimer formation (and, consequently, the fluorescence emission wavelength) is controlled by intramolecular rather than bimolecular interactions. Thus, this probe is highly sensitive to constraints imposed by its environment, and can therefore be used as a viscosity sensor for interior regions of lipoproteins, membranes, micelles, liquid crystals and synthetic polymers.ref Because excimer formation results in a spectral shift (Figure 13.5.2), the probe may be useful for ratio imaging of molecular mobility.ref However, pyrene fatty acids (Fatty Acid Analogs and Phospholipids—Section 13.2) appear to be preferable for this purpose because the uptake of 1,3-bis-(1-pyrene)propane by cells is limited.




Nile Red

The phenoxazine dye nile red (N1142, structure) is used to localize and quantitate lipids, particularly neutral lipid droplets within cells.ref It is selective for neutral lipids such as cholesteryl esters ref (and also, therefore, for lipoproteins) and is suitable for staining lysosomal phospholipid inclusions.ref Nile red is almost nonfluorescent in water and other polar solvents but undergoes fluorescence enhancement and large absorption and emission blue shifts in nonpolar environments.ref Its fluorescence enhancement upon binding to proteins is weaker than that produced by its association with lipids ref (Figure 13.5.3). Ligand-binding studies on tubulin and tryptophan synthase ref have exploited the environmental sensitivity of nile red's fluorescence. Nile red has also been used to detect sphingolipids on thin-layer chromatograms ref and to stain proteins after SDS-polyacrylamide gel electrophoresis.ref

Figure 13.5.3
Fluorescence emission spectra of A) 1,8-ANS (A47) and B) nile red (N1142) bound to protein and phospholipid vesicles. Samples comprised 1 µM dye added to 20 µM bovine serum albumin (BSA) or 100 µM dioctadecenoylglycerophosphocholine (DOPC).

Bimane Azide

Bimane azide (B30600, structure) is a small blue-fluorescent photoreactive alkyl azide (excitation/emission maxima ~375/458 nm) for photoaffinity labeling of proteins, potentially including membrane proteins from within the cell membrane. This reactive fluorophore's small size may reduce the likelihood that the label will interfere with the function of the biomolecule, an important advantage for site-selective probes.

LipidTOX Neutral Lipid Stains

Steatosis, the intracellular accumulation of neutral lipids as lipid droplets or globules, is often triggered by drugs that affect the metabolism of fatty acids or neutral lipids. LipidTOX neutral lipid stains were developed to characterize the effects of drugs and other compounds on lipid metabolism in mammalian cell lines. LipidTOX neutral lipid stains have an extremely high affinity for neutral lipid droplets. These reagents are added after cell fixation and do not require subsequent wash steps after incubation with the sample. Key advantages of this series of neutral lipid stains over conventional stains such as Nile Red include their ready-to-use formulations, their flexibility for multiplexing protocols and their compatibility with LipidTOX phospholipid stains (H34350, H34351; Fatty Acid Analogs and Phospholipids—Section 13.2).

LipidTOX neutral lipid stains are available with green, red and deep red fluorescence emission:

  • HCS LipidTOX Green neutral lipid stain (H34475), with excitation/emission maxima ~495/505 nm (Figure 13.5.4)
  • HCS LipidTOX Red neutral lipid stain (H34476), with excitation/emission maxima ~577/609 nm
  • HCS LipidTOX Deep Red neutral lipid stain (H34477), with excitation/emission maxima ~637/655 nm

These HCS LipidTOX neutral lipid stains have been used to image intracellular lipid accumulation in rat cortical neurons, COS-7 cells and hepatitis C virus (HCV)–infected FT3-7 human hepatoma cells.ref HCS LipidTOX Red neutral lipid stain was used to detect RNAi knockdown of acyl-coenzyme A:cholesterol acyl transferase, isoform 1 (ACAT-1), an endoplasmic reticulum enzyme that regulates the equilibrium between free cholesterol and cholesteryl esters in cells.ref

LipidTOX Green neutral lipid stain is also a component of the HCS LipidTOX Phospholipidosis and Steatosis Detection Kit (H34157, H34158; Fatty Acid Analogs and Phospholipids—Section 13.2), which provides a complete set of reagents for performing high-content screening (HCS) assays to detect and distinguish the intracellular accumulation of phospholipids (phospholipidosis) and of neutral lipids (steatosis) in mammalian cell lines after exposure to test compounds. In addition, HCS LipidTOX neutral lipid stains can be used to monitor the formation and differentiation of adipocytes, a process called adipogenesis. Adipogenesis is of acute interest to the biomedical and drug discovery community as it plays an important role in diseases such as obesity, diabetes and atherosclerosis.

HCS LipidTOX neutral lipid stains are designed for fixed–end point workflows in which formaldehyde-fixed cells in microplates are processed, imaged and analyzed. These stains can easily be detected with fluorescence microscopes or HCS readers equipped with standard filter sets.

LipidTOX Green neutral lipid stain and fatty acid–binding protein  


Figure 13.5.4 (FABP4) antibody labeling in adipocytes. Adipocytes differentiated from 3T3-L1 mouse fibroblasts were fixed with formaldehyde and permeabilized with saponin before labeling with rabbit anti–fatty acid binding protein (FABP4) IgG (red). These cells were then stained with LipidTOX Green neutral lipid stain (H34475, green), counterstained with DAPI (D1306, D21490; blue) and mounted in ProLong Gold antifade reagent (P36930).

Membrane Probes with Environment-Sensitive Spectral Shifts

Prodan and Laurdan

Prodan (P248, structure), introduced by Weber and Farris in 1979, has both electron-donor and electron-acceptor substituent, resulting in a large excited-state dipole moment and extensive solvent polarity–dependent fluorescence shifts ref (Figure 13.5.5). Several variants of the original probe have since been prepared, including the lipophilic derivative laurdan (D250, structure) and thiol-reactive derivatives acrylodan and badan (A433, B6057; Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3), which can be used to confer the environment-sensitive properties of this fluorophore on bioconjugates.

When prodan or its derivatives are incorporated into membranes, their fluorescence spectra are sensitive to the physical state of the surrounding phospholipids.ref In membranes, prodan appears to localize at the surface,ref although Fourier transform infrared (FTIR) measurements indicate some degree of penetration into the lipid interior.ref Excited-state relaxation of prodan is sensitive to the nature of the linkage (ester or ether) between phospholipid hydrocarbon tails and the glycerol backbone.ref In contrast, laurdan's excited-state relaxation is independent of head-group type, and is instead determined by water penetration into the lipid bilayer.ref Two-photon infrared excitation techniques have been successfully applied to both prodan and laurdan, although both probes nominally require ultraviolet excitation ref (~360 nm).

Much experimental work using these probes has sought to characterize coexisting lipid domains based on their distinctive fluorescence spectra,ref an approach that is intrinsically amenable to dual-wavelength ratio measurements.ref Other applications include detecting nonbilayer lipid phases,ref mapping changes in membrane structure induced by cholesterol and alcohols ref and assessing the polarity of lipid/water interfaces.ref Like ANS (see below), prodan is also useful as a noncovalently interacting probe for proteins.ref

Normalized emission spectra of prodan  


Figure 13.5.5 Normalized emission spectra of prodan (P248) excited at 345 nm in 1) cyclohexane, 2) dimethylformamide, 3) ethanol and 4) water.

Dapoxyl Derivative

We have developed a variety of probes based on our Dapoxyl fluorophore.ref Dapoxyl sulfonamide derivatives exhibit UV absorption with maxima near 370 nm, extinction coefficients >24,000 cm-1M-1 and Stokes shifts in excess of 200 nm (spectra). Dapoxyl sulfonic acid (D12800, structure) is an amphiphilic Dapoxyl derivative with generally similar properties and applications to anilinonaphthalene sulfonate (ANS) (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Both ANS and Dapoxyl sulfonic acid have been used in a drug-discovery assay based on the detection of protein thermal denaturation shifts.ref Reactive versions of the Dapoxyl fluorophore are described in Coumarins, Pyrenes and Other Ultraviolet Light-Excitable Fluorophores—Section 1.7 and Derivatization Reagents for Carboxylic Acids and Carboxamides—Section 3.4.

Anilinonaphthalenesulfonate (ANS) and Related Derivatives

The use of anilinonaphthalene sulfonates (ANS) as fluorescent probes dates back to the pioneering work of Weber in the 1950s, and this class of probes remains valuable for studying both membrane surfaces and proteins. Slavik's 1982 review of its properties is recommended reading, especially for the extensive compilation of spectral data.ref The primary member of this class, 1,8-ANS (A47, structure), and its analogs 2,6-ANS (A50) and 2,6-TNS (T53) are all essentially nonfluorescent in water, only becoming appreciably fluorescent when bound to membranes (quantum yields ~0.25) or proteins (quantum yields ~0.7) ref (Figure 13.5.3). This property makes them sensitive indicators of protein folding, conformational changes ref and other processes that modify the exposure of the probe to water (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1). Fluorescence of 2,6-ANS is also enhanced by cyclodextrins, permitting a sensitive method for separating and analyzing cyclodextrins with capillary electrophoresis.ref


Bis-ANS (B153, structure) is superior to 1,8-ANS as a probe for nonpolar cavities in proteins, often binding with an affinity that is orders-of-magnitude higher.ref Bis-ANS has particularly high affinity for nucleotide-binding sites of some proteins.ref It is also useful as a structural probe for tubulin ref and as an inhibitor of microtubule assembly.ref Covalent photoincorporation of bis-ANS into proteins has been reported.ref


The styrene derivative DCVJ (D3923, structure) is a sensitive indicator of tubulin assembly and actin polymerization.ref The fluorescence quantum yield of DCVJ is strongly dependent on environmental rigidity, resulting in large fluorescence increases when the dye binds to antibodies ref and when it is compressed in synthetic polymers or phospholipid membrane interiors.ref DCVJ has been used for microviscosity measurements of phospholipid bilayers.ref

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A47 icon icon 299.34 L pH >6, DMF 372 7800 480 MeOH 1
A50 icon 299.34 L DMF 319 27,000 422 MeOH 1
B153 icon 672.85 L pH >6 395 23,000 500 MeOH 1, 2
B311 icon 444.57 L MeCN, CHCl3 344 80,000 378 MeOH 3
B3932 icon 448.32 F,L DMSO, CHCl3 665 161,000 676 MeOH 4
C34556 icon icon 438.25 F,D,L DMSO 588 68,000 616 MeOH 5
D109 icon 529.63 L DMSO, EtOH 495 85,000 518 MeOH 6
D202 icon 232.32 L DMF, MeCN 350 88,000 452 MeOH 7, 8
D250 icon 353.55 L DMF, MeCN 364 20,000 497 MeOH 9
D3921 icon 248.08 F,L EtOH, DMSO 502 98,000 510 MeOH 4
D3922 icon 262.11 F,L EtOH, DMSO 493 89,000 504 MeOH 4
D3923 icon 249.31 L DMF, DMSO 456 61,000 493 MeOH  
D12800 icon 366.37 L DMSO, H2O 358 25,000 517 MeOH 10
F3857 icon 584.79 L DMSO, EtOH 504 95,000 525 MeOH 6
H110 icon 585.74 L DMSO, EtOH 497 92,000 519 MeOH 6
H34475 icon ~300 F,L DMSO 495 94,000 505 MeOH 5, 13
H34476 icon ~400 F,L DMSO 574 62,000 609 MeOH 5, 13
H34477 icon ~350 F,L DMSO 626 68,000 648 MeOH 5, 13
N1142 icon icon 318.37 L DMF, DMSO 552 45,000 636 MeOH 11
O246 icon 731.50 F,DD,L DMSO, EtOH 556 125,000 578 MeOH 12
P248 icon 227.31 L DMF, MeCN 363 19,000 497 MeOH 9
T53 icon 335.35 L DMF 318 26,000 443 MeOH 1
T204 icon 461.62 D,L DMF, DMSO 355 75,000 430 MeOH 7
  1. Fluorescence quantum yields of ANS and its derivatives are environment dependent and are particularly sensitive to the presence of water. QY of A47 is about 0.4 in EtOH, 0.2 in MeOH and 0.004 in water. Em is also somewhat solvent dependent.ref
  2. B153 is soluble in water at 0.1–1.0 mM after heating.
  3. Absorption spectra of bis-pyrenyl alkanes have additional peaks at ~325 nm and <300 nm. Emission spectra include both monomer (~380 nm and ~400 nm) and excimer (~470 nm) peaks.
  4. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  6. Spectra of this product are pH dependent. Data listed are for basic solutions prepared in methanol containing a trace of KOH.
  8. Stock solutions of DPH (D202) are often prepared in in tetrahydrofuran (THF). Long-term storage of THF solutions is not recommended because of possible peroxide formation in that solvent.
  9. The emission spectrum of P248 is solvent dependent. Em = 401 nm in cyclohexane, 440 nm in CHCl3, 462 nm in MeCN, 496 nm in EtOH and 531 nm in H2O.ref Abs is only slightly solvent dependent. The emission spectra of D250 in these solvents are similar to those of P248.
  10. Em = 520 nm when bound to phospholipid bilayer membranes. Fluorescence in H2O is weak (Em ~600 nm).
  11. The absorption and fluorescence spectra and fluorescence quantum yield of N1142 are highly solvent dependent.ref
  12. This product is intrinsically a sticky gum at room temperature.
  13. Abs/Em in trioctanoylglycerol = 498/507 nm, 582/616 nm and 635/652 nm for H34475, H34476 and H34477, respectively.