The study of lipid organization and dynamics in cellular membranes is a field of active research (1
). In many cases, fluorescent lipid analogs, which mimic the amphiphilic structure of naturally occurring lipids through a polar fluorescent headgroup linked to long hydrocarbon tails, are used to study lipid organization in cellular and model lipid membranes (5
). For example, fluorescent lipid analogs such as DiI or Texas Red dipalmitoyl-phosphatidylethanolamine have been shown to preferentially partition into different lipid phases in bilayers, allowing relatively stable lipid domains with diameters larger than the diffraction limit (~300 nm) to be distinguished(6
). A second strategy is to use environmentally sensitive fluorophores such as laurdan, prodan, dansyl, NBD and the recently reported di-4-ANEPPHQ. These molecules have also been used to label lipid membranes through preferential partitioning into different lipid phases, and offer emission spectra, fluorescence lifetimes or fluorescence quantum yields that vary based on the polarity or the viscosity of the environment(8
). Experiments using environmentally sensitive fluorophores have focused on high concentration imaging, where the membrane is homogeneously labeled and information on a large population of fluorophores is obtained.
With the advent of single-molecule spectroscopy (11
) and its extension to cellular studies (13
), individual lipids and membrane-associated proteins have been followed to characterize the nanoscale local structure within the plasma membrane. In many of these studies, a fluorophore label such as Cy3 (15
) or Cy5 (16
) is covalently linked to the protein or lipid of interest. While this allows single copies of these membrane-associated molecules to be visualized, the fluorescence signal from these fluorophores is relatively stable and not typically used to report on changes in the local environment. Moreover, none of the environmentally sensitive analogs described above have been demonstrated to be useful in single-molecule imaging in the cellular environment. For example, laurdan and prodan photobleach easily and require short excitation wavelengths such as 360 nm, making them difficult to detect at the single-molecule level above the autofluorescence of cells without two-photon excitation. (8
) Thus, the development of an environmentally sensitive fluorescent lipid analog that can be visualized at the single molecule level would enable researchers to probe the local environment that surrounds each single molecule directly, and possibly permit the detection of unstable, nanometer-scale regions or domains.
A new class of fluorophores that possesses a lipid-like amphiphilic structure and is well-suited for single-molecule studies has been reported(19
). These molecules, termed DCDHFs, consist of an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, styrene, etc.). The DCDHFs have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground state dipole moment, and sensitivity to local environment. Structural modifications of the DCDHF fluorophores have tuned the absorption/emission of these molecules from UV to IR wavelengths while retaining their amphiphilic nature. (19
) Moreover, the emission wavelength and fluorescence quantum yield of molecules in this class are sensitive to solvent polarity and local rigidity (19
). For example, in polymer films, high photostabilities (photobleaching quantum yield from 7.5 × 10−7
to 14 × 10−7
) and high fluorescence quantum yields (0.39-0.95) have been reported, while the fluorescence quantum yields for the same DCDHFs in solution are significantly lower (20
). Thus, the DCDHF molecules are a promising new class of environmentally sensitive fluorescent lipid analogs for single-molecule cellular imaging.
In this study, we utilize the seven specific DCDHF lipid analogs shown in as reporters of mobility in the plasma membrane of Chinese hamster ovary (CHO) cells, and describe single-molecule diffusion measurements by recording time-dependent positional trajectories. Single molecules of the DCDHF-N-12 derivative provide total numbers of photons and signal-to-noise on a par with the conventional lipid analog Tritc-DHPE. Future work will be directed toward measuring potential spectral shifts and/or changes in the lifetime or fluorescence quantum yield of the DCDHFs in the cell membrane.
Figure 1 DCDHF derivatives (A) Structures of DCDHF derivatives used in this study and their names. (B) Normalized absorption spectra for the DCDHF derivatives shown in A. (C) Normalized emission spectra for the DCDHF derivatives shown in A. The excitation wavelengths (more ...)