Biology subjects

Dorlhiac, G.

Publications and source records attributed to Dorlhiac, G..

3 recordsLinked to original sources

bioRxiv2021

Multimodal measurements of phototoxicity in nonlinear optical microscopy

Nonlinear optical imaging modalities, such as two-photon microscopy and stimulated Raman scattering (SRS) microscopy, make use of pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we investigated the short and long-term effects of pulsed laser induced phototoxicity. We used bulk RNA sequencing, quantitative measurement of cell proliferation, and measurement of the generation of reactive oxygen species (ROS) to assess phototoxic effects, at different time scales, for a range of laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant ROS generation, differential gene expression, or change in proliferation rates of mouse Neuro2A cells. Changes in proliferation rate and ROS generation were observed under imaging conditions with an excitation intensity of over 600 mW/m2. Repeated imaging of the same field of view at this excitation intensity of over 600 mW/m2 resulted in visual damage to N2A cells. Laser induced perturbations in live cells may impact downstream measurements of cell state including subsequent imaging or molecular measurements. This study provides guidance for imaging parameters that minimize photo-induced perturbations in SRS microscopy to ensure accurate interpretation of experiments with time-lapse imaging or with paired measurements of imaging and sequencing on the same cells.

bioengineering↗

bioRxiv2020

Paper-Thin Multilayer Microfluidic Devices with Integrated Valves

Integrated valve microfluidics has an unparalleled capability to automate the rapid delivery of fluids at the nanoliter scale for high-throughput biological experimentation. However, multilayer soft lithography, which is used to fabricate valve-microfluidics, produces devices with a minimum thickness of around five millimeters. This form-factor limitation prevents the use of such devices in experiments with limited sample thickness tolerance such as 4-pi microscopy, stimulated Raman scattering microscopy, and many forms of optical or magnetic tweezer applications. We present a new generation of integrated valve microfluidic devices that are less than 300 m thick, including the cover-glass substrate, that resolves the thickness limitation. This "thin-chip" was fabricated through a novel soft-lithography technique that produces on-chip micro-valves with the same functionality and reliability of traditional thick valve-microfluidic devices despite the orders of magnitude reduction in thickness. We demonstrated the advantage of using our thin-chip over traditional thick devices to automate fluid control while imaging on a high-resolution inverted microscope. First, we demonstrate that the thin-chip provides improved signal to noise when imaging single cells with two-color stimulated Raman scattering (SRS). We then demonstrated how the thin-chip can be used to simultaneously perform on-chip magnetic manipulation of beads and fluorescent imaging. This study reveals the potential of our thin-chip in high-resolution imaging, sorting, and bead capture-based single-cell multi-omics applications.

bioengineering↗

bioRxiv2018

Ultralarge Modulation of Single Wall Carbon Nanotube Fluorescence Mediated by Neuromodulators Adsorbed on Arrays of Oligonucleotide Rings

Non-covalent interactions between single-stranded DNA (ssDNA) oligonucleotides and single wall carbon nanotubes (SWNTs) have provided a unique class of tunable chemistries for a variety of applications. However, mechanistic insight into both the photophysical and intermolecular phenomena underlying their utility is lacking, resulting in obligate heuristic approaches for producing ssDNA-SWNT based technologies. In this work, we present an ultrasensitive \"turn-on\" nanosensor for neuromodulators dopamine and norepinephrine with strong {Delta}F/F0 of up to 3500%, a signal appropriate for in vivo imaging, and uncover the photophysical principles and intermolecular interactions that govern the molecular recognition and fluorescence modulation of this nanosensor synthesized from the non-covalent conjugation of (GT)6 ssDNA strands on SWNTs. The fluorescence modulation of the ssDNA-SWNT conjugate is shown to exhibit remarkable sensitivity to the ssDNA sequence chemistry, length, and surface density, providing a wealth of parameters with which to tune nanosensor dynamic range and strength of fluorescence turn-on. We employ classical and quantum mechanical molecular dynamics simulations to rationalize our experimental findings. Calculations show that (GT)6 ssDNA form ordered loops around SWNT, inducing periodic surface potentials that modulate exciton recombination lifetimes. Further evidence is presented to elucidate how analyte binding modulates SWNT fluorescence. We discuss the implications of our findings for SWNT-based molecular sensing applications.

neuroscience↗