Abstract
Electroporation of neurons, i.e. electric-field induced generation of membrane nanopores to facilitate internalization of molecules, is a classic technique used in basic neuroscience research and recently has been proposed as a promising therapeutic strategy in the area of neuro-oncology. To optimize electroporation parameters, optical techniques capable of delivering time and spatially-resolved information on electroporation pore formation at the nanometer scale would be advantageous. For this purpose we describe here a novel optical method based on second harmonic generation (SHG) microscopy. Due to the nonlinear and coherent nature of SHG, the 3D radiation lobes from stained neuronal membranes are sensitive to the spatial distribution of scatterers in the illuminated patch, and in particular to nanopore formation.We used phase-array analysis to computationally study the SHG signal as a function of nanopore size and nanopore population density and confirmed experimentally, in accordance with previous work, the dependence of nanopore properties on membrane location with respect to the electroporation electric field; higher nanopore densities, lasting < 5 milliseconds, are observed at membrane patches perpendicular to the field whereas lower density is observed at partly tangent locations. Differences between near-anode and near-cathode cell poles are also measured, showing higher pore densities at the anodic pole compared to cathodic pole. This technique is promising for the study of nanopore dynamics in neurons and for the optimization of novel electroporation-based therapeutic approaches.
Highlights
Second Harmonic Generation (SHG) microscopy is a nonlinear optical technique suitable for structural and functional imaging of biological structures such as collagen, muscle and cell membranes [1,2,3,4,5]
The resulting optical signature is modulated by the orientation [7], electrical properties [8] and, most relevant here, the spatial distribution of the scatterers [9]
Prior to electroporation, the nanopore-free membrane can be modeled for the purpose of SHG radiation as a collection of SynaptoredTM C2 scattering molecules, uniformly distributed in space
Summary
Second Harmonic Generation (SHG) microscopy is a nonlinear optical technique suitable for structural and functional imaging of biological structures such as collagen, muscle and cell membranes [1,2,3,4,5]. The resulting optical signature is modulated by the orientation [7], electrical properties [8] and, most relevant here, the spatial distribution of the scatterers [9]. Electropermeabilization or electroporation is a method widely employed for delivery of biomolecules into cells [10,11,12]. A pulsed electric field (duration usually ≈ 1ms) is created by a pair of electrodes, opening transient pores in proximal cell membranes and facilitating passive entry of molecules. Electroporation has received renewed attention because of the development of electrochemotherapy, a new therapeutic approach for highly localized delivery of drugs to a wide range of cancer types [13, 14] including hard to treat neuro-oncology tumors [15]
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