Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites

Abstract

Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of membrane electrophysiology in cells and cellular networks.Recently, it has been shown that the nonlinear optical phenomenon of second harmonic generation (SHG) is a sensitive monitor of cellular membrane potential (1, 2). To observe SHG with sensitivities comparable to linear optical phenomena, tightly focused pulsed laser light with high peak power is required. As reviewed by Nie and Zare (3) single molecules were successfully observed in linear optical imaging as early as 1981 and most recently, with great success, by using confocal microscopy to limit the out-of-focus light that severely degrades the signal to noise in linear optical imaging. However, SHG, either in an imaging or a nonimaging mode, has not been accomplished with single molecule sensitivity.SHG arises from the second term of the expansion of the molecular electron polarizability shown below and thus is called a second-order nonlinear optical phenomenon.where P is the polarization, E is the applied optical electric field, and (n) are the nth order optical susceptibilities. In the recent past, the third term of this equation has generated a lot of interest in biological imaging because it is responsible for the nonlinear optical phenomenon of two-photon fluorescence (TPF) (4-8). The use of nonlinear optics results in naturally high x, y, and z spatial resolution, and, in general with nonlinear optical microscopy, photodamage and photobleaching are reduced because the out-of-focus light does not result in excitation. However, for the particular case of SHG, no photochemistry occurs even in the focal plane because the signal, stimulated by nonresonant radiation, does not involve an excited state with a finite lifetime. Optical effects arising from the first term (linear processes) and the third terms of this equation have no fundamental restriction on the symmetric distribution of the dye molecules that give rise to these phenomena in the cells. Second harmonic generation, on the other hand, has a requirement that symmetrically distributed chromophores will not contribute to the observed signal. This symmetry restriction results in a considerable advantage to the nonlinear optical imaging of cell membranes and their membrane potential because only those molecules that are asymmetrically distributed in the cell membrane contribute to the observe...

Biogenic Synthesis of Nanoparticles and Potential Applications: An Eco- Friendly Approach

“…Currently, the majority imaging studies using gold nanoparticles are carried out in cell culture [90]. The functional cellular imaging about single molecules has been reported by Peleg et al [91], captivating benefit of the enhanced second harmonic signal by antibody conjugated gold nanospheres.…”

Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects

“…Resonant energy transfer (RET) systems consisting of organic dye molecules and noble metal nanoparticles have recently gained considerable interest in biophotonics [1][2][3][4] as well as in materials science [5,6]. Closely related are donor-acceptor pairs of organic dye molecules forming Förster resonant energy transfer (FRET) systems.…”

Tip-enhanced nano-Raman analytical imaging of locally induced strain distribution in carbon nanotubes

“…It covers a wide range of applications including nanoscale circuits [1][2][3] , cancer therapy 4 , holography 5 , efficient solar cells 6 , metamaterial devices [7][8][9] , single-molecule detection 10,11 , biosensing 12 and nanoresolution imaging 13 . Some of the functions of surface plasmon polaritons utilized in plasmonic techniques are field enhancement, photon nanoconfinement and spectral tunability.…”