ABSTRACT
To circumvent this problem, one can apply the second-order nonlinear optical method of secondharmonic generation (SHG) to optically measure the membrane potentials. We will not review the theoretical basis of SHG, which is covered in the chapters in Part I of this volume, but will briey provide some pointers to its essential features. In SHG, incident light at frequency ω generates light at 2ω on interacting with the neuron, which selectively images membranes, and not bulk regions because of symmetry requirements [7]. erefore, by cancelation of the dipole moments of the chromophores, there is essentially no SHG signal from inside or outside the cell. Meanwhile, the plasma membrane contributes to position the SHG chromophores in the same orientation, either by lipophilic adsorption to the membrane leaet, or by electrostatic forces. Moreover, if the SHG chromophore is delivered only to one side of the membrane (either extra or intracellularly), the plasma membrane then acts as a symmetry-breaking interface, generates therefore an array of oriented chromophores with a dipole asymmetry, which is an ideal circumstance for strong SHG. is means that when one measures SHG from neurons there is essentially no background SHG signal and all SHG photons are generated in the plasma membrane, exactly where the electrical eld is located. is physicochemical “perfect storm” has not passed unnoticed by investigators: Indeed, in the last decade, following the pioneering work of Lewis and Loew [8], a number of groups, including ourselves, have successfully applied SHG and performed high-resolution optical measurements of membrane potential from living neurons [9-15].
