Classic Loew Lab ANEP VSDs

di-4-ANEPPS, di-8-ANEPPS, di-4-ANEPPDHQ, di-3-ANEPPDHQ,  di-2-ANEPEQ, di-1-ANEPEQ

The ANEP class of dyes were originally developed 35 years ago (1-3) and have stood the test of time by serving to meet the voltage imaging and recording needs of the research community. They respond to voltage changes by an electrochromic mechanism (4-6) that assures the speed needed for high-fidelity recording of action potentials (7). This mechanism also lends itself to many different applications, allowing the chromophore to be incorporated into a variety of VSDs with varying solubility and hydrophobicity. While many newer VSDs with improved sensitivity and photostability are available from Potentiometric Probes, we feel it is important to make these “classic” dyes available so that researchers can continue to use them for their established experimental protocols.

Chemical structure diagram of a di-n-ANEPX compound with variations for X as PS, PDHQ, and EQ groups.

Di-8-ANEPPS (1, 8-10)

This is a more hydrophobic version of Di-4-ANEPPS with essentially the same voltage sensitivity. For this reason, Di-8-ANEPPS is more resistant to washout. Interestingly, it is also more resistant to internalization and therefore allows for longer-term experiments. It has been used for dual-wavelength ratio imaging in both excitation and emission modes. Finally, it has become the dye of choice for investigations of membrane dipole potential.

Di-4-ANEPPDHQ & Di-3-ANEPPDHQ (11-13)

These are more hydrophilic versions of the ANEP class of VSDs and can penetrate more deeply into tissue. This has made them excellent choices for 2-photon imaging of brain slices. They have also been shown to be sensitive to lipid environments and can be used to study membrane domains and lipid rafts.

Classic VSDs originally synthesized by Rina Hildesheim in the laboratory of Amiram Grinvald; RH1691 and RH482

Developed in Amiram Grinvald's neuroscience laboratory at the Weizmann Institute, these VSDs were employed in some of the first in vivo mammalian recordings (21-23) and are still used by many labs today. These VSDs are mostly unavailable from other sources and are offered by us as a service to the optical recording research community.

Di-4-ANEPPS (1-3)

This VSD is arguably the most versatile and widely used fluorescent dye for optical voltage recording and imaging in both cardiac and neuronal systems. It is offered by many specialty suppliers of fluorescent probes, but why not obtain it from the original source? We supply it at highly competitive pricing. In addition, we offer user support to help you design your experiment or assay. Additionally, should this classic VSD prove inadequate for your preparation or assay, we can use the results of your initial studies to offer advice on optimizing your measurement and the best choice of a more advanced VSD.

Di-2-ANEPEQ & Di-1-ANEPEQ (aka JPW1114 & JPW3028) (14-18)

Short alkyl chains (ethyl and methyl, respectively) render these doubly positively charged VSDs highly water soluble. These VSDs are primarily used for intracellular recording or imaging of single cells in a brain slice or in vivo. This is achieved by allowing them to diffuse out of a patch pipette in the whole cell patch configuration so that only the patched cell is stained. The high aqueous solubility of the dyes makes it possible to make the staining solution in the patch sufficiently concentrated to stain and then record voltage from long axons, dendritic arbors, and dendritic spines. Although Di-2-ANEPEQ and Di-1-ANEPEQ are routinely used by many labs, we believe the newer fluorinated dye ElectroFluor530s™ (di-2-AN(F)EPPTEA) (19, 20) is superior in sensitivity and photochemical stability.  

    1. Loew, L. M., L. B. Cohen, J. Dix, E. N. Fluhler, V. Montana, G. Salama, and J. Y. Wu. 1992. A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. The Journal of membrane biology 130:1-10. Pubmed

    2. Fluhler, E., V. G. Burnham, and L. M. Loew. 1985. Spectra, membrane binding, and potentiometric responses of new charge shift probes. Biochemistry 24:5749-5755. Pubmed

    3. Hassner, A., D. Birnbaum, and L. M. Loew. 1984. Charge-shift probes of membrane potential. Synthesis. The Journal of Organic Chemistry 49:2546-2551 DOI

    4. Loew, L. M., G. W. Bonneville, and J. Surow. 1978. Charge shift optical probes of membrane potential. Theory. Biochemistry 17:4065-4071. Pubmed

    5. Loew, L. M., S. Scully, L. Simpson, and A. S. Waggoner. 1979. Evidence for a charge-shift electrochromic mechanism in a probe of membrane potential. Nature 281:497-499. Pubmed

    6. Loew, L. M., and L. L. Simpson. 1981. Charge-shift probes of membrane potential: a probable electrochromic mechanism for p-aminostyrylpyridinium probes on a hemispherical lipid bilayer. Biophysical journal 34:353-365.1327479. Pubmed

    7. Loew, L. M., L. B. Cohen, B. M. Salzberg, A. L. Obaid, and F. Bezanilla. 1985. Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon. Biophysical journal 47:71-77.1435075. Pubmed

    8. Bedlack, R. S., Jr., M. D. Wei, S. H. Fox, E. Gross, and L. M. Loew. 1994. Distinct electric potentials in soma and neurite membranes. Neuron 13:1187-1193. Pubmed

    9. Gross, E., R. S. Bedlack, Jr., and L. M. Loew. 1994. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential. Biophysical journal 67:208-216.1225351. Pubmed

    10. Bullen, A., and P. Saggau. 1999. High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes. Biophysical journal 76:2272-2287. Pubmed

    11. Obaid, A. L., L. M. Loew, J. P. Wuskell, and B. M. Salzberg. 2004. Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179-190. Pubmed

    12. Fisher, J. A. N., J. R. Barchi, C. G. Welle, G.-H. Kim, P. Kosterin, A. L. Obaid, A. G. Yodh, D. Contreras, and B. M. Salzberg. 2008. Two-Photon Excitation of Potentiometric Probes Enables Optical Recording of Action Potentials From Mammalian Nerve Terminals In Situ. J Neurophysiol 99:1545-1553. Pubmed

    13. Jin, L., A. C. Millard, J. P. Wuskell, X. Dong, D. Wu, H. A. Clark, and L. M. Loew. 2006. Characterization and application of a new optical probe for membrane lipid domains. Biophysical journal 90:2563-2575.1403187. Pubmed

    14. Antic, S., and D. Zecevic. 1995. Optical signals from neurons with internally applied voltage-sensitive dyes. Journal of Neuroscience 15:1392-1405. Pubmed

    15. Zecevic, D. 1996. Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes. Nature 381:322-325 Pubmed

    16. Antic, S., J. P. Wuskell, L. Loew, and D. Zecevic. 2000. Functional profile of the giant metacerebral neuron of Helix aspersa: temporal and spatial dynamics of electrical activity in situ. J Physiol 527 Pt 1:55-69.2270048. Pubmed

    17. Foust, A., M. Popovic, D. Zecevic, and D. A. McCormick. 2010. Action Potentials Initiate in the Axon Initial Segment and Propagate through Axon Collaterals Reliably in Cerebellar Purkinje Neurons. The Journal of Neuroscience 30:6891-6902. Pubmed

    18. Zhou, W. L., P. Yan, J. P. Wuskell, L. M. Loew, and S. D. Antic. 2007. Intracellular long-wavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. J Neurosci Methods 164:225-239.2001318. Pubmed

    19. Acker, C. D., P. Yan, and L. M. Loew. 2011. Single-voxel recording of voltage transients in dendritic spines. Biophysical journal 101:L11-L13. Pubmed

    20. Acker, C. D., E. Hoyos, and L. M. Loew. 2016. EPSPs Measured in Proximal Dendritic Spines of Cortical Pyramidal Neurons. eNeuro, 3(2). Pubmed

    21. Shoham, D., D. E. Glaser, A. Arieli, T. Kenet, C. Wijnbergen, Y. Toledo, R. Hildesheim, and A. Grinvald. 1999. Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage-sensitive dyes. Neuron. 24(4):791-802. Pubmed

    22. Grinvald, A., and R. Hildesheim. 2004. VSDI: A new era in functional imaging of cortical dynamics. Nat Rev Neurosci. 5(11):874-885. Pubmed

    23. Grinvald, A., D. B. Omer, D. Sharon, I. Vanzetta, and R. Hildesheim. 2016. Voltage-Sensitive Dye Imaging of Neocortical Activity. Cold Spring Harb Protoc. 2016(1):pdb top089367. Pubmed

    24. Grinvald, A., A. Fine, I. C. Farber, and R. Hildesheim. 1983. Fluorescence Monitoring of Electrical Responses from Small Neurons and Their Processes. Biophysical Journal. 42:195-198. Pubmed