Sherazade Aknoun

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.

Quantitative phase imaging applied to laser damage detection and analysis

We investigate phase imaging as a measurement method for laser damage detection and analysis of laser-induced modification of optical materials. Experiments have been conducted with a wavefront sensor based on lateral shearing interferometry associated with a high-magnification optical microscope. The system has been used for the in-line observation of optical thin films and bulk samples, laser irradiated in two different conditions: 500 fs pulses at 343 and 1030 nm, and millisecond to second irradiation with a CO2 laser at 10.6 μm. We investigate the measurement of the laser-induced damage threshold of optical material by detection and phase changes and show that the technique realizes high sensitivity with different optical path measurements lower than 1 nm. Additionally, the quantitative information on the refractive index or surface modification of the samples under test that is provided by the system has been compared to classical metrology instruments used for laser damage or laser ablation characterization (an atomic force microscope, a differential interference contrast microscope, and an optical surface profiler). An accurate in-line measurement of the morphology of laser-ablated sites, from few nanometers to hundred microns in depth, is shown.

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

Abstract. 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.

Tomographic incoherent phase imaging, a diffraction tomography alternative for any white-light microscope

In this paper, we discuss the possibility of making tomographic reconstruction of the refractive index of a microscopic sample using a quadriwave lateral shearing interferometer, under incoherent illumination. A Z-stack is performed and the acquired incoherent elecromagnetic fields are deconvoluted before to retrieve in a quantitative manner the refractive index. The results are presented on polystyrene beads and can easily be expanded to biological samples. This technique is suitable to any white-light microscope equipped with nanometric Z-stack module.

Quadriwave lateral shearing interferometry as a quantification tool for microscopy. Application to dry mass determination of living cells, temperature mapping, and vibrational phase imaging

A Quadri-Wave Lateral Shearing Interferometer (QWLSI) is an efficient tool for measuring phase gradients of optical beams along two perpendicular directions. Post-processing integration then allows obtaining the complete phase spatial distribution of the beam. By placing a QWLSI on the exit image plane of such a microscope, we are able to measure the complex field spatial distribution in this plane, and then to retrieve the quantitative optical path difference (OPD) of the observed sample. Here, we demonstrate that we can extend the technique to new applications, were different physical phenomena produce a given sample-induced change in the phase of the exit optical beam that modulates the incident wavefront. More precisely, we used direct refractive-induced OPD, thermal-induced OPD, and resonant vibrational-induced OPD to produce phase contrast images of living cells, temperature distribution of complex patterns of nanostructures, and Raman spectra of polystyrene beads, respectively. In the case of refractive-induced OPD of living cells, we also show that the OPD distribution of a living cell can be used to monitor its dry mass during the cell cycle.

Quantitative birefringence imaging of biological samples using quadri-wave interferometry

We describe a new technique based on the use of a high-resolution quadri-wave lateral shearing interferometry wave front sensor to perform quantitative linear birefringence measurements on biological samples. The system combines quantitative phase images with different excitation polarizations to create birefringence images. This technique is fast and compatible with living samples. It gives information about the local retardance and structure of their anisotropic components.

Versatile quantitative phase imaging system applied to high-speed, low noise and multimodal imaging (Conference Presentation)

Quadriwave lateral shearing interferometry (QWLSI) is a well-established quantitative phase imaging (QPI) technique based on the analysis of interference patterns of four diffraction orders by an optical grating set in front of an array detector [1]. As a QPI modality, this is a non-invasive imaging technique which allow to measure the optical path difference (OPD) of semi-transparent samples. We present a system enabling QWLSI with high-performance sCMOS cameras [2] and apply it to perform high-speed imaging, low noise as well as multimodal imaging. This modified QWLSI system contains a versatile optomechanical device which images the optical grating near the detector plane. Such a device is coupled with any kind of camera by varying its magnification. In this paper, we study the use of a sCMOS Zyla5.5 camera from Andor along with our modified QWLSI system. We will present high-speed live cell imaging, up to 200Hz frame rate, in order to follow intracellular fast motions while measuring the quantitative phase information. The structural and density information extracted from the OPD signal is complementary to the specific and localized fluorescence signal [2]. In addition, QPI detects cells even when the fluorophore is not expressed. This is very useful to follow a protein expression with time. The 10 µm spatial pixel resolution of our modified QWLSI associated to the high sensitivity of the Zyla5.5 enabling to perform high quality fluorescence imaging, we have carried out multimodal imaging revealing fine structures cells, like actin filaments, merged with the morphological information of the phase. References [1]. P. Bon, G. Maucort, B. Wattellier, and S. Monneret, “Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells,” Opt. Express, vol. 17, pp. 13080–13094, 2009. [2] P. Bon, S. Lécart, E. Fort and S. Lévêque-Fort, “Fast label-free cytoskeletal network imaging in living mammalian cells,” Biophysical journal, 106(8), pp. 1588-1595, 2014

Label-free three dimensional reconstruction of biological samples(Conference Presentation)

We describe the use of spatially incoherent illumination combined with quantitative phase imaging (QPI) [1] to make tridimensional reconstruction of semi-transparent biological samples. Quantitative phase imaging is commonly used with coherent illumination for the relatively simple interpretation of the phase measurement. We propose to use spatially incoherent illumination which is known to increase lateral and axial resolution compared to classical coherent illumination. The goal is to image thick samples with intracellular resolution [2]. The 3D volume is imaged by axially scanning the sample with a quadri-wave lateral shearing interferometer used as a conventional camera while using spatially incoherent white-light illumination (native microscope halogen source) or NIR light. We use a non-modified inverted microscope equipped with a Z-axis piezo stage. A z-stack is recorded by objective translation along the optical axis. The main advantages of this approach are its easy implementation, compared to the other state-of-the-art diffraction tomographic setups, and its speed which makes even label-free 3D living sample imaging possible. A deconvolution algorithm is used to compensate for the loss in contrast due to spatially incoherent illumination. This makes the tomographic volume phase values quantitative. Hence refractive index could be recovered from the optical slices. We will present tomographic reconstruction of cells, thick fixed tissue of few tens of micrometers using white light, and the use of NIR light to reach deeper planes in the tissue.

In-line quantitative phase imaging for damage detection and analysis

We investigate quantitative phase imaging as a measurement method for laser damage detection and analysis of laser induced modification of optical materials. Experiments have been conducted with a wavefront sensor based on lateral shearing interferometry technique associated to a high magnification optical microscope. The system has been used for in situ observation of optical thin films and bulk samples irradiated by 500fs pulses. It is shown that the technique realizes high sensitivity, convenient use and can provide quantitative information on the refractive index or surface modification of the samples under test.