Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sickling ß-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle ß-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous ß-globin gene or regions involved in fetal hemoglobin silencing. Transfection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.

Photothermal Control of Heat-Shock Protein Expression at the Single Cell Level.

Laser heating of individual cells in culture recently led to seminal studies in cell poration, fusion, migration, or nanosurgery, although measuring the local temperature increase in such experiments remains a challenge. Here, the laser-induced dynamical control of the heat-shock response is demonstrated at the single cell level, enabled by the use of light-absorbing gold nanoparticles as nanosources of heat and a temperature mapping technique based on quadriwave lateral shearing interferometry (QLSI) measurements. As it is label-free, this approach does not suffer from artifacts inherent to previously reported fluorescence-based temperature-mapping techniques and enables the use of any standard fluorescent labels to monitor in parallel the cell's response.

Phase measurement of a segmented wave front using PISton and TILt interferometry (PISTIL).

New architectures for telescopes or powerful lasers require segmented wave front metrology. This paper deals with a new interferometric wave front sensing technique called PISTIL (PISton and TILt), able to recover both piston and tilts of segment beams. The main advantages of the PISTIL technique are the absence of a reference arm and an access to the tilt information. An explanation of the principle, as well as an experimental implementation and the use of a segmented active mirror, are presented. Measurement errors of λ/200 for piston and 40 µrad for tilts have been achieved, well beyond performances requested for the above mentioned applications.

Quantitative retardance imaging of biological samples using quadriwave lateral shearing interferometry.

We describe a new technique based on the use of a high-resolution quadri-wave lateral shearing interferometer to perform quantitative linear retardance and birefringence measurements on biological samples. The system combines quantitative phase images with varying polarization excitation to create retardance images. This technique is compatible with living samples and gives information about the local retardance and structure of their anisotropic components. We applied our approach to collagen fibers leading to a birefringence value of (3.4 ± 0.3) · 10(-3) and to living cells, showing that cytoskeleton can be imaged label-free.

Enhanced 3D spatial resolution in quantitative phase microscopy using spatially incoherent illumination.

We describe the use of spatially incoherent illumination to make quantitative phase imaging of a semi-transparent sample, even out of the paraxial approximation. The image volume electromagnetic field is collected by scanning the image planes with a quadriwave lateral shearing interferometer, while the sample is spatially incoherently illuminated. In comparison to coherent quantitative phase measurements, incoherent illumination enriches the 3D collected spatial frequencies leading to 3D resolution increase (up to a factor 2). The image contrast loss introduced by the incoherent illumination is simulated and used to compensate the measurements. This restores the quantitative value of phase and intensity. Experimental contrast loss compensation and 3D resolution increase is presented using polystyrene and TiO(2) micro-beads. Our approach will be useful to make diffraction tomography reconstruction with a simplified setup.

Modeling quantitative phase image formation under tilted illuminations.

A generalized product-of-convolution model for simulation of quantitative phase microscopy of thick heterogeneous specimen under tilted plane-wave illumination is presented. Actual simulations are checked against a much more time-consuming commercial finite-difference time-domain method. Then modeled data are compared with experimental measurements that were made with a quadriwave lateral shearing interferometer.

Noniterative boundary-artifact-free wavefront reconstruction from its derivatives.

Wavefront sensors are usually based on measuring the wavefront derivatives. The most commonly used approach to quantitatively reconstruct the wavefront uses discrete Fourier transform, which leads to artifacts when phase objects are located at the image borders. We propose here a simple approach to avoid these artifacts based on the duplication and antisymmetrization of the derivatives data, in the derivative direction, before integration. This approach completely erases the border effects by creating continuity and differentiability at the edge of the image. We finally compare this corrected approach to the literature on model images and quantitative phase images of biological microscopic samples, and discuss the effects of the artifacts on the particular application of dry mass measurements.

Quantitative phase microscopy for non-invasive live cell population monitoring.

We present here a label-free development based on preexisting Quantitative Phase Imaging (QPI) that allows non-invasive live monitoring of both individual cells and cell populations. Growth, death, effect of toxic compounds are quantified under visible light with a standard inverted microscope. We show that considering the global biomass of a cell population is a more robust and accurate method to assess its growth parameters in comparison to compiling individually segmented cells. This is especially true for confluent conditions. This method expands the use of light microscopy in answering biological questions concerning live cell populations even at high density. In contrast to labeling or lysis of cells this method does not alter the cells and could be useful in high-throughput screening and toxicity studies.

Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells.

Phase imaging with a high-resolution wavefront sensor is considered. This is based on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope. The measurement technology is explained both in the scope of wave optics and geometrical optics in order to discuss its implementation on a conventional microscope. In particular we consider the effect of a non spatially coherent source on the phase-image signal-to-noise ratio. Precise measurements of the phase-shift introduced by microscopic beads or giant unilamellar vesicles validate the principle and show the accuracy of the methods. Diffraction limited images of living COS-7 cells are then presented, with a particular focus on the membrane and organelle dynamics.

Microscale Temperature Shaping Using Spatial Light Modulation on Gold Nanoparticles.

Heating on the microscale using focused lasers gave rise to recent applications, e.g., in biomedicine, biology and microfluidics, especially using gold nanoparticles as efficient nanoabsorbers of light. However, such an approach naturally leads to nonuniform, Gaussian-like temperature distributions due to the diffusive nature of heat. Here, we report on an experimental means to generate arbitrary distributions of temperature profiles on the micrometric scale (e.g. uniform, linear, parabolic, etc) consisting in illuminating a uniform gold nanoparticle distribution on a planar substrate using spatially contrasted laser beams, shaped using a spatial light modulator (SLM). We explain how to compute the light pattern and the SLM interferogram to achieve the desired temperature distribution, and demonstrate the approach by carrying out temperature measurements using quantitative wavefront sensing.

Living cell dry mass measurement using quantitative phase imaging with quadriwave lateral shearing interferometry: an accuracy and sensitivity discussion.

Single-cell dry mass measurement is used in biology to follow cell cycle, to address effects of drugs, or to investigate cell metabolism. Quantitative phase imaging technique with quadriwave lateral shearing interferometry (QWLSI) allows measuring cell dry mass. The technique is very simple to set up, as it is integrated in a camera-like instrument. It simply plugs onto a standard microscope and uses a white light illumination source. Its working principle is first explained, from image acquisition to automated segmentation algorithm and dry mass quantification. Metrology of the whole process, including its sensitivity, repeatability, reliability, sources of error, over different kinds of samples and under different experimental conditions, is developed. We show that there is no influence of magnification or spatial light coherence on dry mass measurement; effect of defocus is more critical but can be calibrated. As a consequence, QWLSI is a well-suited technique for fast, simple, and reliable cell dry mass study, especially for live cells.

Optical detection and measurement of living cell morphometric features with single-shot quantitative phase microscopy.

We present a quadriwave lateral shearing interferometer used as a wavefront sensor and mounted on a commercial non-modified transmission white-light microscope as a quantitative phase imaging technique. The setup is designed to simultaneously make measurements with both quantitative transmission phase and fluorescence modes: phase enables enhanced contrasted visualization of the cell structure including intracellular organelles, while fluorescence allows a complete and precise identification of each component. After the characterization of the phase measurement reliability and sensitivity on calibrated samples, we use these two imaging modes to measure the characteristic optical path difference between subcellular elements (mitochondria, actin fibers, and vesicles) and cell medium, and demonstrate that phase-only information should be sufficient to identify some organelles without any labeling, like lysosomes. Proof of principle results show that the technique could be used either as a qualitative tool for the control of cells before an experiment, or for quantitative studies on morphology, behavior, and dynamics of cells or cellular components.

Optimization of the dynamic wavefront control of a pulsed kilojoule/nanosecond-petawatt laser facility.

The wavefront aberrations in a large-scale, flash-lamp-pumped, high-energy, high-power glass laser system can degrade considerably the quality of the final focal spot, and limit severely the repetition rate. The various aberrations induced on the Laboratoire pour l'Utilisation des Lasers Intenses (LULI), laser facility (LULI2000) throughout the amplification are identified and analyzed in detail. Based on these analyses, an optimized procedure for dynamic wavefront control is then designed and implemented. The lower-order Zernike aberrations can be effectively reduced by combining an adaptive-optics setup, comprising a bimorph deformable mirror and a four-wave lateral shearing interferometer, with a precise alignment system. This enables the laser chain to produce a reproducible focal spot close to the diffraction limit (Strehl ratio approximately 0.7). This allows also to increase the repetition rate, initially limited by the recovery time of the laser amplifiers, by a factor of 2 (one shot per hour). The proposed procedure provides an attractive alternative for dynamic correction of the wavefront aberrations of a laser facility as complex as the LULI2000.

Optical switching and contrast enhancement in intense laser systems by cascaded optical parametric amplification.

Optical parametric chirped-pulse amplification (OPCPA) can be used to improve the prepulse contrast in chirped-pulse amplification systems by amplifying the main pulse with a total saturated OPCPA gain, while not affecting the preceding prepulses of the seed oscillator mode-locked pulse train. We show that a simple modification of a multistage OPCPA system into a cascaded optical parametric amplifier (COPA) results in an optical switch and extreme contrast enhancement that can completely eliminate the preceding and trailing oscillator pulses. Instrument-limited measurement of a prepulse contrast ratio of 1.4 x 10(11) is demonstrated from COPA at a 30 mJ level.

Piston measurement by quadriwave lateral shearing interferometry.

We present what is to our knowledge a new method for measuring the relative piston between two independent beams separated by a physical gap, typical of petawatt facilities. The feasibility of this measurement, based on quadriwave lateral shearing interferometry, has been demonstrated experimentally: piston has been measured with accuracy and sensitivity better than 50 nm.

Wave-front reconstruction from multidirectional phase derivatives generated by multilateral shearing interferometers.

To increase the accuracy of wave-front evaluation, we propose to exploit the natural capability of multiple lateral shearing interferometers to measure simultaneously more than two orthogonal phase derivatives. We also describe a method, based on Fourier-transform analysis, that uses this multiple information to reconstruct the wave-front under study.