Digital Holographic Imaging for Label-Free Phenotypic Profiling, Cytotoxicity, and Chloride Channels Target Screening

Cellular assays using label-free Digital Holographic Microscopy (DHM) have been previously validated for cell viability assays in a drug screening context. Our automated DHM system allows performing fast and cost-effective screening assays for a wide range of applications for monitoring cell morphological changes and cell movements upon interaction with interfering compounds. In addition to these classic phenotypic assays, it has been demonstrated that target-based cellular assays can also be addressed by DHM for therapeutically relevant chloride channel receptors. Our DH-imaging (DHI) technology, potentially scalable for screening by imaging approaches in a high-throughput manner can also deliver highly informative data through long term experiments. Three examples of phenotypic screens are detailed in the present chapter: a label-free profiling approach, a cell proliferation assay, and methods for monitoring the activity of the GABAA chloride channel receptor.

Simultaneous Optical Recording in Multiple Cells by Digital Holographic Microscopy of Chloride Current Associated to Activation of the Ligand-Gated Chloride Channel GABA(A) Receptor

Daniel Boss, Christian Depeursinge, Pascal Jourdain, Pierre Marquet, Corinne Moratal, Benjamin Rappaz

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Public Library of Science2012

Image-Based Marker-Free Screening of GABA(A) Agonists, Antagonists, and Modulators

Damiano Banfi, Pascal Jourdain, Fabien Kuttler, Pierre Marquet, Benjamin Rappaz, Gerardo Turcatti

The ionotropic GABA(A) receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABA(A) receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABA(A) receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABA(A) receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABA(A) receptor and more generally with any ligand-gated ion channels and transporters.

2019

Passive and active ion permeation through lipid bilayers investigated by second harmonic water imaging

Maksim Eremchev

Lipid membranes are complex and dynamic systems which are known to mediate signaling processes between cells and their environment. To do this multiple ion channels and pumps are involved in controlling the in- and out-flux of various ions (K+, Na+, Mg2+, Ca2+, etc.) and help maintain and regulate a concentration gradient of ions across the membrane. Additionally, membranes are semi-permeable for some species including water and even ions. However, molecular level information about passive and active transport of ions across lipid membranes is either missing or incomplete and ignores membrane hydration without which a membrane would not self-assemble.In this project, we use high-throughput wide-field second harmonic (SH) microscopy to learn about the molecular level structure of membrane interfaces during those processes by imaging the non-resonant response of interfacial water. We show that this technique is extremely sensitive to the transmembrane distribution of interfacial ions which disturb membrane hydration and thus change SH contrast. We apply this technique to probe the molecular structure(orientational order of water, strength of ion binding) at the interfaces of freestanding lipid bilayers and giant unilamellar vesicles (GUVs). We start with SH imaging of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by an aqueous solution on either side. We observe a change in SH intensity upon channel activation that is traced back to a change in the orientational distribution of water molecules caused by transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. Later on, in order to improve SH contrast from low-emitting samples (such as interfacial water) and thus be able to study even more fundamental and delicate surface processes, we develop and build a femtosecond optical parametric amplifier. This laser system generates a tunable output in the range from 670 nm to 1000 nm at a 1 MHz repetition rate with a minimum pulse duration of 23 fs at 900 nm. Comparing our current femtosecond laser system with the new one at equal fluences we experimentally observe a 30 times improvement in SH contrast of low-emitting samples.Then, we present a new approach of label-free second harmonic imaging of GUV hydration which directly reports on the cross-membrane distribution of divalent cations bound to negatively charged lipid headgroups. We use this property to study passive transport of Ca2+ ions through lipid membranes. By varying the hydrophobic core of the bilayer, we observe Ca2+ translocation for mono-unsaturated (DOPC:DOPA) membranes which is reduced upon adding cholesterol. A complete inhibition of translocation is observed for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. In order to gain more insight into the mechanism of divalent cation transport, we perform a series of experiments with different ions (Mg2+, Ba2+, Ca2+and Cu2+). Surprisingly, we observe translocation of Ba2+, Ca2+ and Cu2+ ions through unsaturated DOPC:DOPA membranes which fully neutralize the inner membrane leaflet. Translocation time increases in the order Cu2+< Ca2+< Ba2+, while Mg2+ ions do not show any permeation. The observed trend in passive transport correlates with a trend in ion binding strength measured on impermeable GUVs with the same headgroup composition.

EPFL2021

Passive and active ion permeation through lipid bilayers investigated by second harmonic water imaging

Maksim Eremchev

EPFL2021