Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4

Page Contents

• Dialkylcarbocyanine Probes
• Dialkylaminostyryl Probes
• Data Table
• Ordering Information

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).

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 and long-term labeling of cells in culture (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) and oxacarbocyanines (D275) 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 (FAST DiO, D3898; FAST DiI, D3899, D7756)
• DiI and DiO analogs with shorter alkyl tails (DiIC₁₂(3), D383; DiIC₁₆(3), D384)
• Long-wavelength light–excitable carbocyanines (DiD, D307, D7757)
• Infrared light–excitable carbocyanine (DiR, D12731)
• 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 DiIC_n(3) probes have absorption and fluorescence spectra compatible with rhodamine (TRITC) optical filter sets, whereas DiOC_n(3) analogs can be used with fluorescein (FITC) optical filter sets. 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. Their fluorescence in water is quite weak. 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. Dialkylcarbocyanines are also exceptionally photostable.

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). Their extinction coefficients are somewhat larger and fluorescence quantum yields much larger than those of carbocyanines such as DiI. Moreover, photoexcitation of DiD seems to cause less collateral damage than photoexcitation of DiI in live cells. The DiIC₁₈(7) tricarbocyanine probe (DiR, D12731) has excitation and emission in the infrared, which may make the dye useful as an in vivo tracer for labeled cells and liposomes in live organisms.

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)
• Anionic sulfophenyl derivatives of DiI and DiO  (SP-DiIC₁₈(3), D7777; SP-DiOC₁₈(3), D7778)
• Sulfonate derivatives of DiI and DiD (DiIC₁₈(3)-DS; DiIC₁₈(5)-DS, D12730)

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. 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.

DiI and DiO as Probes of Membrane Structure

The orientation of DiIC₁₈(3) in membranes has been determined by fluorescence polarization microscopy. 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. The lateral partitioning behavior of dialkylindocarbocyanines in membranes has been investigated by fluorescence recovery after photobleaching (FRAP), calorimetry, lifetime measurements  and fluorescence resonance energy transfer techniques  (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  (~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, as well as fusion of liposomes with planar bilayers. Membrane fusion can also be measured by fluorescence resonance energy transfer to DiIC₁₈(3) from dansyl- or NBD-labeled phospholipid donors  or by direct imaging. 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. Energy transfer from DiIC₁₈(5) to DiIC₁₈(7) should be similarly effective. Lipophilic carbocyanines have also been used to elicit photosensitized destabilization of liposomes, to sensitize photoaffinity labeling of the viral glycoprotein hemagglutinin, to image membrane domains in lipid monolayers  and to develop a fiber-optic potassium sensor.

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. Their lateral diffusion coefficients in isolated fluid- and gel-phase bilayers are independent of the carbocyanine alkyl chain length. 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. Combined lateral diffusion measurements of labeled proteins and lipids have demonstrated that transformed  and permeabilized  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  and around IgE receptor complexes. 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)  (D384).

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The lipophilic aminostyryl probes 4-Di-10-ASP, DiA (4-Di-16-ASP, D3883) and FAST DiA insert in membranes with their two alkyl tails and their fluorophore oriented parallel to the phospholipid acyl chains (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. 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; however, the fluorescence spectrum of DiA is very broad, with appreciable intensity from about 510 nm to 690 nm. 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, the diunsaturated analog of DiA, is intended to facilitate these studies by accelerating dye diffusion within the membrane.

The FM 1-43, 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.

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