Time-Resolved Molecular Transport across Living Cell Membranes

Abstract

It is shown that the nonlinear optical phenomenon known as second-harmonic generation can be used for label-free, time-resolved study of the transport of molecules through living cell membranes. The adsorption and transport of a 300-Da molecular-mass hydrophobic ion at the Escherichia coli membrane is observed. Remarkably, at low ion concentrations, the second-harmonic generation technique clearly exposes a multistep molecular transport process: Transport of the molecular ion across the outer and cytoplasmic membranes of the Gram-negative bacteria is recorded, in sequence, in time. Fitting of the data to a multiprocess kinematic model reveals that the transport of this hydrophobic ion through the outer membrane is much faster than through the cytoplasmic membrane, likely reflecting the effectiveness of ion transport porins. The observations illustrate an experimental means for studying the interactions of small molecules with cell membranes.

Adsorption and transport of charged vs. neutral hydrophobic molecules at the membrane of murine erythroleukemia (MEL) cells

“…30,000 ± 5000 counts/s), the time-resolved SHG signal is invariant after the initial jump following the introduction of MG into the cell suspension. As compared to prior examples of molecular uptake into living cells [17,18], the absence of decay in the SHG signal following the initial rise implies that MG does not traverse the cell membrane. Specifically, if MG was able to transport across the cell membrane and adsorb onto the inner leaflet of the membrane, the anisotropically ordered molecules adsorbed on the two opposing leaflets of the membrane would have opposite orientations due to the symmetry of the bilayer structure.…”

“…The experimental setup for measuring the SH signal is similar to that for studying SH light scattering from other colloidal systems; the details of which have been reported previously [17,18,43]. In general, a fast pulsed laser source with a high peak power is necessary for inducing SHG, in which the low pulse energy ensures that laser-induced heating of the sample is kept to a minimum.…”

“…It has previously been demonstrated that the nonlinear optical technique, second harmonic generation (SHG), can be used to study time-resolved molecular adsorption onto and transport across membranes of living biological cells [17,18]. In this approach, an SHG signal is detected at twice the frequency (i.e., 2ω) of an incident fundamental light source of frequency ω. SHG is induced through dipole allowed polarizability, which does not exist in molecules exhibiting inversion symmetry (i.e., centrosymmetric).…”

“…In addition to probing adsorption and transport across living cell membranes [17,18,20], SHG has been applied to other biologically relevant systems including liposomes [21][22][23][24][25], neurons [26], and collagen networks [27,28].…”

Investigating Two-Photon-Induced Fluorescence in Rhodamine-6G in Presence of Cetyl-Trimethyl-Ammonium-Bromide

“…Such a possibility has been recently realized when second-harmonic generation (SHG) was employed to probe the transport phenomenon across membrane [42]. This nonlinear optical response in terms of fluorescence enhancement in presence of surfactant can find significant application in the fields of membrane dynamics as compared to bulk dynamics and specific chromophorelipid interactions wherein lipids can act as surfactant.…”