Fast-Response Probes—Section 22.2

Page Contents

• ANEP Dyes
• RH Dyes
• FRET-Pair Membrane Potential Sensors
• Data Table
• Ordering Information

Our fast-response potential-sensitive probes (see Figure 22.1.1A in Introduction to Potentiometric Probes—Section 22.1) are listed in Characteristics and selected applications of Molecular Probes fast-response probes—Table 22.1, along with their charges, optical responses and selected applications.

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. Zwitterionic 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 22.2.1). 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 22.2.1). Some other applications are listed in Characteristics and selected applications of Molecular Probes fast-response probes—Table 22.1.

Figure 22.2.1 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 (2000) 279:H1421.

Cationic ANEP Dyes

In collaboration with Leslie Loew and Joe Wuskell of the University of Connecticut, we offer a series of potential-sensitive cationic 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-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.

Originally synthesized by Rina Hildesheim, the RH dyes include an extensive series of dialkylaminophenylpolyenylpyridinium dyes that are principally used for functional imaging of neurons (Characteristics and selected applications of Molecular Probes fast-response probes—Table 22.1). The existence of numerous RH dye analogs reflects the observation that no single dye provides the optimal response under all experimental conditions. Currently, the most widely used RH dyes are RH 237 (S1109, ), RH 414 (T1111, ), RH 421 (S1108, ) and RH 795 (R649, ). Physiological effects of staining with different analogs are not equivalent. For example, staining of the cortex with RH 414 causes arterial constriction, whereas staining with RH 795 does not. RH 795 produced negligible side effects when tested in vitro using hippocampal slices and in vivo using single-unit recordings in cat and monkey visual cortices. Electrophysiological measurements indicate a broadening of action potentials that is attributable to the staining of cultured neurons with RH 237.

Like the ANEP dyes, the RH dyes exhibit varying degrees of fluorescence excitation and emission spectral shifts in response to membrane potential changes. Their absorption and fluorescence spectra are also strongly dependent on the environment. Spectra of RH 414 bound to phospholipid vesicles are similar to those obtained on neuronal plasma membranes. Using the RH dyes in conjunction with fluorescent Ca²⁺ indicators allowed the simultaneous optical mapping of membrane potential (with RH 237) and intracellular calcium (with rhod-2 AM; R1245MP, R1244; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) in cardiomyocyte monolayers; rhod-FF (R23983, Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) was used to check for buffering of calcium dynamics by the high-affinity rhod-2 indicator.

Fluorescence resonance energy transfer (FRET) between a mobile lipophilic anion in the membrane interior and a static donor fluorophore on the membrane surface provides a potential-sensing mechanism that generates a more sensitive fluorescence response than electrochromic dyes and a more rapid temporal response than intracellular–extracellular ion translocation. A particularly effective implementation of this concept uses DiOC₁₈(3) or DiOC₁₆(3) (D275, V22886, D1125; Tracers for Membrane Labeling—Section 14.4) as the static reference marker in combination with the mobile anion dipicrylamine. Characterization of this approach by Bradley and co-workers demonstrated depolarization-induced fluorescence changes of >50% per 100 mV with submillisecond time constants in whole-cell patch-clamped HEK 293 cells. In neuronal cultures and brain slices, action potentials generated fluorescence increases relative to the resting baseline signal of >25% per 100 mV.