The dyes in this section are all amphiphilic probes—molecules that comprise a charged fluorophore that localizes the probe at the membrane's surface and lipophilic aliphatic "tails" that insert into the membrane and thus anchor the probe to the membrane. In addition to labeling model membranes, most of these probes are very useful for cell tracing applications (Tracers for Membrane Labeling—Section 14.4). Summary of our lipophilic carbocyanine and aminostyryl tracers—Table 14.3 lists all of our lipophilic carbocyanine and aminostyryl tracers and compares their properties and uses. Our FM dyes, which are also amphiphilic styryl dyes but with less lipophilic character than the dyes in this section, are particularly useful for labeling membranes of live cells and for following synaptosome recycling (Probes for Following Receptor Binding and Phagocytosis—Section 16.1).

Dialkylcarbocyanine Probes

Carbocyanines are among the most strongly light-absorbing dyes known and have proven to be useful tools in several different areas of research. Carbocyanines with short alkyl tails attached to the imine nitrogens are employed both as membrane-potential sensors (Slow-Response Probes—Section 22.3) and as organelle stains for mitochondria and the endoplasmic reticulum (Probes for Mitochondria—Section 12.2, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4). Those with longer alkyl tails (≥12 carbons) have an overall lipophilic character that makes them useful for neuronal tracing ref and long-term labeling of cells in culture ref (Tracers for Membrane Labeling—Section 14.4), as well as for noncovalent labeling of lipoproteins (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). This section describes the use and properties of dialkylcarbocyanines as general-purpose probes of membrane structure and dynamics.

DiI, DiO, DiD, DiR and Analogs

The most widely used carbocyanine membrane probes have been the octadecyl (C18) indocarbocyanines (D282, D3911; structure) and oxacarbocyanines (D275, structure) often referred to by the generic acronyms DiI and DiO, or more specifically as DiIC18(3) and DiOC18(3), where the subscript is the number of carbon atoms in each alkyl tail and the bracketed numeral is the number of carbon atoms in the bridge between the indoline or benzoxazole ring systems. We also offer several variations on these basic structures (Tracers for Membrane Labeling—Section 14.4, Summary of our lipophilic carbocyanine and aminostyryl tracers—Table 14.3):

  • DiI and DiO analogs with unsaturated alkyl tails (Δ9-DiI, D3886; FAST DiO, D3898; FAST DiI, D3899, D7756)
  • DiI and DiO analogs with shorter alkyl tails (DiIC12(3), D383; structure; DiIC16(3), D384; DiOC16(3), D1125)
  • Long-wavelength light–excitable carbocyanines (DiD, D307, D7757; structure)
  • Infrared light–excitable carbocyanine (DiR, D12731; structure)
  • Chloromethylbenzamido DiI and sulfonated DiI and DiO derivatives (see below)

Spectral Properties of Dialkylcarbocyanines

The spectral properties of dialkylcarbocyanines are largely independent of the lengths of the alkyl chains, and are instead determined by the heteroatoms in the terminal ring systems and the length of the connecting bridge. The DiICn(3) probes have absorption and fluorescence spectra compatible with rhodamine (TRITC) optical filter sets (spectra), whereas DiOCn(3) analogs can be used with fluorescein (FITC) optical filter sets (spectra). The emission maxima of DiIC18(3) and DiOC18(3) incorporated in dioctadecenoylphosphocholine (dioleoyl PC or DOPC) liposomes (Figure 13.4.1) are similar to those of the dyes in methanol.

The very large molar extinction coefficients of carbocyanine fluorophores are their most outstanding spectral property. Their fluorescence quantum yields are only modest—about 0.07 for DiI in methanol and about three-times greater in amphiphilic solvents such as octanol.ref Their fluorescence in water is quite weak.ref The excited-state lifetimes of carbocyanine fluorophores in lipid environments are short (~1 nanosecond), which is an advantage for flow cytometry applications because it allows more excitation/de-excitation cycles during flow transit; the overall decay is multi-exponential.ref Dialkylcarbocyanines are also exceptionally photostable.ref

The red He-Ne laser–excitable indodicarbocyanines such as DiD (DiIC18(5); D307, D7757) have long-wavelength absorption and red emission (Figure 13.4.1, spectra). Their extinction coefficients are somewhat larger and fluorescence quantum yields much larger than those of carbocyanines such as DiI.ref Moreover, photoexcitation of DiD seems to cause less collateral damage than photoexcitation of DiI in live cells.ref The DiIC18(7) tricarbocyanine probe (DiR, D12731) has excitation and emission in the infrared (spectra), which may make the dye useful as an in vivo tracer for labeled cells and liposomes in live organisms.ref



Figure 13.4.1 Normalized fluorescence emission spectra of DiO (D275), DiI (D282), DiD (D307) and DiR (D12731) bound to phospholipid bilayer membranes.

Substituted DiI and DiO Derivatives

We have synthesized various derivatives of DiI, DiO and DiD. All of these derivatives have octadecyl (C18) tails identical to those of DiI (D282, D3911) and DiO (D275), thereby preserving the excellent membrane retention characteristics of the parent molecules. A variety of substitutions have been made on the indoline or benzoxazole ring systems:

  • Chloromethylbenzamido DiI derivatives (CellTracker CM-DiI; C7000, C7001; structure)
  • Anionic sulfophenyl derivatives of DiI and DiO ref (SP-DiIC18(3), D7777, structure; SP-DiOC18(3), D7778, structure)
  • Sulfonate derivatives of DiI and DiD (DiIC18(3)-DS, D7776, structure; DiIC18(5)-DS, D12730, structure)

Although these derivatives have primarily been developed to provide improved fixation and labeling in long-term cell tracing applications (Tracers for Membrane Labeling—Section 14.4), they also offer several features that can potentially be exploited for investigating membrane structure and dynamics. For researchers wishing to carry out comparative evaluations, our Lipophilic Tracer Sampler Kit (L7781) provides 1 mg samples of each of nine different carbocyanine derivatives, including several of the newer substituted derivatives:

  • DiI (DiIC18(3))
  • DiD (DiIC18(5))
  • DiR (DiIC18(7))
  • DiO (DiOC18(3))
  • DiA (4-Di-16-ASP)
  • DiIC18(3)-DS
  • SP-DiIC18(3)
  • SP-DiOC18(3)
  • 5,5'-Ph2-DiIC18(3)

The fluorescence quantum yields of the sulfophenyl and phenyl derivatives (measured in methanol) are generally two- to threefold greater than those of DiI and DiO. In particular, we have found that the sulfophenyl derivatives (SP-DiIC18(3), D7777; SP-DiOC18(3), D7778) bound to phospholipid model membranes have approximately fivefold higher quantum yields than DiI and DiO. DiIC18(5)-DS (D12730) has been used in combination with an NBD-labeled glycerophosphoserine probe in a novel resonance energy transfer assay that detects inner monolayer membrane hemifusion, avoiding erroneous indications of membrane fusion due to lipid mixing and other environmental effects in the outer monolayer.ref The negative charge and greater water solubility of the sulfonated carbocyanines results in modified lateral and transverse distributions of these probes in lipid bilayers relative to those of DiI and DiO. This characteristic has been exploited to identify plasma membrane lipid domains that are responsive to electrical stimulation of outer hair cells in the inner ear.ref

DiI and DiO as Probes of Membrane Structure

The orientation of DiIC18(3) in membranes has been determined by fluorescence polarization microscopy.ref The long axis of the fluorophore is parallel to the membrane surface, and the two alkyl chains protrude perpendicularly into the lipid interior (Figure 13.2.1D in Fatty Acid Analogs and Phospholipids—Section 13.2). There are conflicting reports in the literature regarding the ease of transbilayer migration ("flip-flop") of lipophilic indocarbocyanines.ref The lateral partitioning behavior of dialkylindocarbocyanines in membranes has been investigated by fluorescence recovery after photobleaching (FRAP),ref calorimetry,ref lifetime measurements ref and fluorescence resonance energy transfer techniques ref (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). These studies demonstrate that the probe distribution between coexisting fluid and gel phases depends on the similarity of the alkyl chain lengths of the probe and the lipid. In general, the more dissimilar the lengths, the greater the preference for fluid-phase over gel-phase lipids. For example, the shorter-chain DiIC12(3) has a substantial preference for the fluid phase (~6:1) in DOPC, whereas DiIC18(3) is predominantly distributed in the gel phase ref (~1:10). Consequently, long-chain dialkylcarbocyanines are among the best probes for detecting particularly rigid gel phases.

Lipophilic carbocyanines have been used to visualize membrane fusion and cell permeabilization that occurs in response to electric fields,ref as well as fusion of liposomes with planar bilayers.ref Membrane fusion can also be measured by fluorescence resonance energy transfer to DiIC18(3) from dansyl- or NBD-labeled phospholipid donors ref or by direct imaging.ref In Langmuir–Blodgett films, excited-state energy transfer from DiIC18(3) to DiIC18(5) is exceptionally efficient because of the favorable orientations of the fluorophores.ref Energy transfer from DiIC18(5) to DiIC18(7) should be similarly effective. Lipophilic carbocyanines have also been used to elicit photosensitized destabilization of liposomes,ref to sensitize photoaffinity labeling of the viral glycoprotein hemagglutinin,ref to image membrane domains in lipid monolayers ref and to develop a fiber-optic potassium sensor.ref

DiI and DiO as Probes of Membrane Dynamics

Despite their reasonably good photostability, dialkylcarbocyanines are widely employed to measure lateral diffusion processes using fluorescence recovery after photobleaching (FRAP) techniques.ref Their lateral diffusion coefficients in isolated fluid- and gel-phase bilayers are independent of the carbocyanine alkyl chain length.ref Phase-separated populations of lipophilic carbocyanine dyes can be distinguished by their diffusion rates and can therefore be used to define lateral domains in cell membranes.ref Combined lateral diffusion measurements of labeled proteins and lipids have demonstrated that transformed ref and permeabilized ref cells show marked changes in protein diffusion whereas lipid diffusion rates remain unchanged. In other cases, coupling of lipid and protein mobility has been identified in the form of relatively immobilized lipid domains in yeast plasma membranes ref and around IgE receptor complexes.ref A different photobleaching technique, which depends on the absence of diffusional fluorescence recovery, was employed to determine lipid flow direction in locomoting cells by following the movement of a photobleached stripe of DiIC16(3) ref (D384).

Dialkylaminostyryl Probes

The lipophilic aminostyryl probes 4-Di-10-ASP (D291, structure), DiA (4-Di-16-ASP, D3883; structure) and FAST DiA (D7758, structure) insert in membranes with their two alkyl tails and their fluorophore oriented parallel to the phospholipid acyl chains ref (Figure 13.2.1H in Fatty Acid Analogs and Phospholipids—Section 13.2). When these dialkylaminostyryl probes bind to membranes, they exhibit a strong fluorescence enhancement; their fluorescence in water is minimal. The interfacial solvation of the aminostyryl fluorophore causes a large blue shift of the absorption spectrum of the membrane-bound probe.ref For example, the absorption maximum of DiA is 456 nm when incorporated into DOPC liposomes and 490 nm when in methanol. The fluorescence emission maximum of DiA in the membrane environment is 590 nm, which is quite close to that observed for probes with shorter alkyl tails such as 4-Di-10-ASP;ref however, the fluorescence spectrum of DiA is very broad, with appreciable intensity from about 510 nm to 690 nm (spectra). Consequently, DiA can be detected as green, orange or even red fluorescence, depending on the optical filter employed. Like the lipophilic carbocyanines, DiA is commonly used for neuronal membrane tracing (Tracers for Membrane Labeling—Section 14.4). FAST DiA (D7758), the diunsaturated analog of DiA, is intended to facilitate these studies by accelerating dye diffusion within the membrane.

The FM 1-43 (structure), FM 1-43FX, FM 4-64 and FM 5-95 dyes, which are discussed in detail in Probes for Following Receptor Binding and Phagocytosis—Section 16.1, are styryl dyes that also exhibit high Stokes shifts and broad fluorescence emission but have less lipophilic character than the 4-Di-10-ASP and DiA probes. The FM dyes are commonly used to define the outer membranes of liposomes and live cells and to detect synaptosome recycling.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
C7000 icon icon 1051.50 F,D,L DMSO, EtOH 553 134,000 570 MeOH  
C7001 icon icon 1051.50 F,D,L DMSO, EtOH 553 134,000 570 MeOH  
D275 icon icon 881.72 L DMSO, DMF 484 154,000 501 MeOH  
D282 icon icon 933.88 L DMSO, EtOH 549 148,000 565 MeOH  
D291 icon icon 618.73 L DMSO, EtOH 492 53,000 612 MeOH 1
D307 icon icon 959.92 L DMSO, EtOH 644 260,000 665 MeOH 2
D383 icon icon 765.56 L DMSO, EtOH 549 144,000 565 MeOH 3
D384 icon icon 877.77 L DMSO, EtOH 549 148,000 565 MeOH  
D1125 icon icon 825.61 L DMSO, DMF 484 156,000 501 MeOH  
D3883 icon icon 787.05 L DMSO, EtOH 491 52,000 613 MeOH 1
D3886 icon icon 925.49 F,L,AA DMSO, EtOH 549 144,000 564 MeOH 2
D3898 icon icon 873.65 F,L,AA DMSO, DMF 484 138,000 499 MeOH  
D3899 icon icon 925.82 F,L,AA DMSO, EtOH 549 143,000 564 MeOH 2
D3911 icon icon 933.88 L DMSO, EtOH 549 148,000 565 MeOH  
D7756 icon icon 1017.97 F,L,AA DMSO, EtOH 549 148,000 564 MeOH  
D7757 icon icon 1052.08 L DMSO, EtOH 644 193,000 663 MeOH  
D7758 icon icon 899.80 F,L,AA DMSO, EtOH 492 41,000 612 MeOH 1
D7776 icon 993.54 L DMSO, EtOH 555 144,000 570 MeOH  
D7777 icon 1145.73 L DMSO, EtOH 556 164,000 573 MeOH  
D7778 icon 1115.55 L DMSO, EtOH 497 175,000 513 MeOH  
D12730 icon 1019.58 L DMSO, EtOH 650 247,000 670 MeOH  
D12731 icon icon 1013.41 L DMSO, EtOH 748 270,000 780 MeOH  
  1. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The difference is typically 20 nm for absorption and 80 nm for emission, but varies considerably from one dye to another. Styryl dyes are generally nonfluorescent in water.
  2. This product is intrinsically a liquid or an oil at room temperature.
  3. This product is intrinsically a sticky gum at room temperature.
For Research Use Only. Not for use in diagnostic procedures.