Di-4-ANEPPS and Di-8-ANEPPS
The ANEP (AminoNaphthylEthenylPyridinium) dyes developed by Leslie Loew and colleagues 
are among the most sensitive of the fast-response probes. Di-4-ANEPPS (D1199, 
) and di-8-ANEPPS (D3167, ) exhibit fairly uniform 10% per 100 mV changes in fluorescence intensity in a variety of tissue, cell and model membrane systems. The millisecond-range temporal characteristics of the ANEP dyes compensate for this modest response amplitude (Figure 22.4). Di-4-ANEPPS is internalized in the cell rather rapidly, precluding its use in all but very short-term experiments, whereas di-8-ANEPPS is better retained in the outer leaflet of the plasma membrane. In addition, although both ANEP dyes exhibit good photostability and low toxicity, di-8-ANEPPS is reported to be slightly more photostable and significantly less phototoxic than di-4-ANEPPS.
Like other styryl dyes, the ANEP dyes are essentially nonfluorescent in aqueous solutions and exhibit spectral properties that are strongly dependent on their environment. When bound to phospholipid vesicles, di-8-ANEPPS has absorption/emission maxima of ~467/631 nm (
), as compared with ~498/713 nm in methanol. The fluorescence excitation/emission maxima of di-4-ANEPPS bound to neuronal membranes are ~475/617 nm.
Both di-4-ANEPPS and di-8-ANEPPS respond to increases in membrane potential (hyperpolarization) with a decrease in fluorescence excited at approximately 440 nm and an increase in fluorescence excited at 530 nm. These spectral shifts permit the use of ratiometric methods (Loading and Calibration of Intracellular Ion Indicators—Note 19.1) to correlate the change in fluorescence signal with membrane potential. Using di-8-ANEPPS, Loew and colleagues were able to follow changes in membrane potential along the surface of a single mouse neuroblastoma cell in their study of the mechanisms underlying cathode-directed neurite elongation and to define differences between transmembrane potentials of neurites and somata. Potential-dependent fluorescence emission ratio measurements (ratio of emission intensities at 560 nm and 620 nm following excitation at 475 nm) have also been reported using both di-4-ANEPPS and di-8-ANEPPS (Figure 22.4). Some other applications are listed in Characteristics and selected applications of Molecular Probes' fast-response probes—Table 22.1.
Figure 22.4 Detection of action potentials in intact rabbit hearts using the fast potentiometric probe di-4-ANEPPS (
D1199). Excised rabbit hearts were loaded with di-4-ANEPPS by perfusion with dye-containing medium. Fluorescence was excited at 488 nm by an argon-ion laser. Emission components at 540 ± 6 nm (green) and >610 nm (red) were detected simultaneously by two photomultipliers (panel A). The ratio of the green to red signals (panel B) displayed a larger fractional change during action potential cycles than either of the component signals; it also followed transmembrane voltage contours recorded simultaneously by an intracellular microelectrode (panel C). In addition, fluorescence ratio measurements reduce the motion artifacts that typically distort optical signals detected from contracting hearts. Figure reproduced with permission from Am J Physiol Heart Circ Physiol 279, H1421 (2000).
Other ANEP Dyes
In collaboration with Leslie Loew and Joe Wuskell of the University of Connecticut, Molecular Probes offers a series of five potential-sensitive ANEP dyes. The water-soluble di-2-ANEPEQ (JPW 1114, D6923; ) can be either microinjected into cells, a mode of delivery that intensifies the staining of remote neuronal processes, or applied topically to deeply stain brain tissue. Microinjection of di-2-ANEPEQ into neurons in ganglia of the snail Helix aspersa produced an approximately 50-fold improvement in voltage-sensitive signals from distal processes over that obtained with conventional absorption- and fluorescence-based staining methods. Di-12-ANEPPQ (D6927, ) is useful for potential-sensitive retrograde labeling of neurons using techniques similar to those employed for lipophilic carbocyanine and aminostyryl tracers (Tracers for Membrane Labeling—Section 14.4). Di-1-ANEPIA (D36800, ) has been coupled to the cysteines in a voltage-gated potassium channel and then used to monitor localized electric field changes during the gating process with submillisecond resolution. Di-3-ANEPPDHQ (D36801, ) and di-4-ANEPPDHQ (D36802, ) both exhibit very low rates of internalization and good signal-to-noise ratios and are useful for neural network analysis. Di-4-ANEPPDHQ has proven useful for visualizing cholesterol-enriched lipid domains in model membranes.
