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Research

Project IV - Technology Development For Studies of Cellular Regulation
Aim 3: Improving second harmonic based membrane potential imaging

We are developing a novel method to increase cell viability during membrane potential imaging using membrane dye SHG. The dramatic increase in signal-to-noise obtained by using membrane SHG signals rather than fluorescence is significant, but as in all fast membrane potential imaging experiments, cell viability is a critical issue once the illumination is turned on.   The method described here should help eliminate the light-induced damage and make membrane SHG imaging more viable than fluorescence based methods. Currently, mV precision measurements can be made with patch electrodes at one or two localities on single cells, or they can be imaged with low precision at extended sites by using fluorescence probes that change in intensity by only a few percent per 100 mV. The use of nonlinear microscopy with second harmonic (SH) generation by membrane localized styryl dyes has been shown to be sensitive to membrane potential. In cell monolayers, this relative signal change per 100 mV has been shown to reach nearly an order of magnitude larger than that seen from fluorescence probes (Campagnola et al. 1999). We have investigated dyes synthesized and supplied to us by Leslie Loew (University of Connecticut Health Center), Seth Marder and Joseph Perry (then at the University of Arizona) and M. Blanchard-Desce (Synthèse et Electrosynthèse Organiques, CNRS, Cedex, France) that were designed to have a large SH “effective cross-section” and possibly a large sensitivity to applied electric fields. To identify probes with the largest SH response, we screened these dyes using voltage clamp measurements on cultured cell monolayers. Because second harmonic generation is a spatially coherent optical process, its generated signal increases in proportion to the square of the concentration of cooperating noncentrosymmetric molecules. However, at the high concentration of dyes that provide the best SH signal are not usable in the cell membrane, because of phototoxic effects of illumination of the cells at high dye concentrations, as assessed by voltage clamp recordings. Nonetheless, we were able to demonstrate neural action potential monitoring by SHG in Aplysia neurons using a specifically designed membrane dye (Dombeck et al. 2004), and later in brain slices (Fig. 1) using the commercially available dye FM4-64 (Dombeck et al. 2005). Our initial work in this area has been primarily for proof-of-principle, but we have a number of active collaborators who have projects that would greatly benefit from the ability to measure fast membrane potential changes with higher signal-to-noise ratios than is currently possible.

We have developed a systematic toxicity screening method to judge the harmfulness of the dyes both with and without laser illumination (Sacconi et al. 2006); however, cell damage due to excitation of the dye – regardless of the particular dye – is still the major factor limiting the usefulness of SH membrane potential imaging. We have tried adding anti-oxidants and quenchers with only limited success. However, if we could quench the excited state immediately after the laser pulse (SHG will have already occurred within ~fs), we should be able to eliminate damage caused by the excited state. In order to do this, we propose to quench the excited state using STimulated Emission Depletion (STED), much in the same way that Stefan Hell achieves super-resolution (Hell 2007), only much easier to apply since (1) we will not need to engineer the STED beam PSF, (2) STED beam intensity stability is not as critical, and (3) we are not interested in collecting any fluorescence, only SHG at 530 nm, so we can use the entire FM4-64 emission region for depletion. To quench the excited state we split the beam from our Fianium 1060 nm fiber laser (Fig. 2A), and use a portion of it to generate continuum light using a photonic crystal fiber (Figure 2B) that is mixed back into the light path with a variable delay. We have generated the needed continuum light for depletion of the FM4-64 excited state using a highly non-linear fiber (SC-5.0-1040) from Crystal Fibre (Denmark) as shown in Figure 2C.  The pulsewidth of the continuum light is ~12 ps due to fiber dispersion, which will greatly reduce any unwanted nonlinear effects by the STED pulses.   We are now beginning a set of FM4-64 SH membrane potential imaging experiments using Aplysia neurons and the protocols established in (Sacconi et al. 2006) to establish whether this novel strategy can be used to lessen the phototoxicty of SHG membrane potential imaging.

References

Campagnola, P. J., M. D. Wei, et al. (1999). "High-resolution nonlinear optical imaging of live cells by second harmonic generation." Biophys J 77(6): 3341-9.

Dombeck, D. A., M. Blanchard-Desce, et al. (2004). "Optical recording of action potentials with second-harmonic generation microscopy." J Neurosci 24(4): 999-1003.

Dombeck, D. A., L. Sacconi, et al. (2005). "Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy." J Neurophysiol 94(5): 3628-36.

Hell, S. W. (2007). "Far-field optical nanoscopy." Science 316(5828): 1153-8.

 

 



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