Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy.

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

A Theoretical High-Density Nanoscopy Study Leads to the Design of UNLOC, a Parameter-free Algorithm.

Single-molecule localization microscopy (SMLM) enables the production of high-resolution images by imaging spatially isolated fluorescent particles. Although challenging, the result of SMLM analysis lists the position of individual molecules, leading to a valuable quantification of the stoichiometry and spatial organization of molecular actors. Both the signal/noise ratio and the density (Dframe), i.e., the number of fluorescent particles per µm2 per frame, have previously been identified as determining factors for reaching a given SMLM precision. Establishing a comprehensive theoretical study relying on these two parameters is therefore of central interest to delineate the achievable limits for accurate SMLM observations. Our study reports that in absence of prior knowledge of the signal intensity α, the density effect on particle localization is more prominent than that anticipated from theoretical studies performed at known α. A first limit appears when, under a low-density hypothesis (i.e., one-Gaussian fitting hypothesis), any fluorescent particle distant by less than ∼600 nm from the particle of interest biases its localization. In fact, all particles should be accounted for, even those dimly fluorescent, to ascertain unbiased localization of any surrounding particles. Moreover, even under a high-density hypothesis (i.e., multi-Gaussian fitting hypothesis), a second limit arises because of the impossible distinction of particles located too closely. An increase in Dframe is thus likely to deteriorate the localization precision, the image reconstruction, and more generally the quantification accuracy. Our study firstly provides a density-signal/noise ratio space diagram for use as a guide in data recording toward reaching an achievable SMLM resolution. Additionally, it leads to the identification of the essential requirements for implementing UNLOC, a parameter-free and fast computing algorithm approaching the Cramér-Rao bound for particles at high-density per frame and without any prior knowledge of their intensity. UNLOC is available as an ImageJ plugin.

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.

Laser-induced birefringence measurements by quantitative polarized-phase microscopy.

A technique that provides quantitative and spatially resolved retardance measurement is studied for application to laser-induced modification in transparent materials. The method is based on the measurement of optical path differences between two wavefronts carrying different polarizations, measured by a wavefront sensor placed in the image plane of a microscope. We have applied the technique to the investigation of stress distribution induced by CO2 laser processing of fused silica samples. By comparing experiments to the results of thermomechanical simulations we demonstrate quantitative agreement between measurements and simulations of optical retardance. The technique provides an efficient and simple way to measure retardance of less than 1 nm with a diffraction-limited spatial resolution in transparent samples, and coupled to thermomechanical simulations it gives access to birefringence distribution in the sample.

Ultra-thin rigid endoscope: two-photon imaging through a graded-index multi-mode fiber.

Rigid endoscopes like graded-index (GRIN) lenses are known tools in biological imaging, but it is conceptually difficult to miniaturize them. In this letter, we demonstrate an ultra-thin rigid endoscope with a diameter of only 125 µm. In addition, we identify a domain where two-photon endoscopic imaging with fs-pulse excitation is possible. We validate the ultra-thin rigid endoscope consisting of a few cm of graded-index multi-mode fiber by using it to acquire optically sectioned two-photon fluorescence endoscopic images of three-dimensional samples.

Time-resolved quantitative-phase microscopy of laser-material interactions using a wavefront sensor.

We report on a simple and efficient technique based on a wavefront sensor to obtain time-resolved amplitude and phase images of laser-material interactions. The main interest of the technique is to obtain quantitative self-calibrated phase measurements in one shot at the femtosecond time-scale, with high spatial resolution. The technique is used for direct observation and quantitative measurement of the Kerr effect in a fused silica substrate and free electron generation by photo-ionization processes in an optical coating.

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.

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.

Independent synchronized control and visualization of interactions between living cells and organisms.

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode's surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.

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.

Two-photon lensless endoscope.

We report a first demonstration of two-photon endoscopic imaging with a lensless endoscope. The endoscope probe is a double-clad bundle of single-mode fibers; point excitation and scanning is achieved by coherent combining of femtosecond light pulses propagating in the single-mode fibers; and back-scattered two-photon signal is collected through the multi-mode inner cladding. We demonstrate the two-photon endoscope on a test sample of rhodamine 6G crystals.

Toward endoscopes with no distal optics: video-rate scanning microscopy through a fiber bundle.

We report a step toward scanning endomicroscopy without distal optics. The focusing of the beam at the distal end of a fiber bundle is achieved by imposing a parabolic phase profile across the exit face with the aid of a spatial light modulator. We achieve video-rate images by galvanometric scanning of the phase tilt at the proximal end. The approach is made possible by the bundle, designed to have very low coupling between cores.

Wide-field vibrational phase imaging in an extremely folded box-CARS scattering geometry.

We present a method that allows one to measure the real and imaginary parts of the third-order susceptibility in a wide-field coherent anti-Stokes Raman scattering setup using a quadriwave lateral shearing interferometer. This permits the retrieval of the undistorted Raman spectrum and the removal of a nonresonant signal from the surrounding solvent, which otherwise may overwhelm weak resonances.

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.

Imaging the Gouy phase shift in photonic jets with a wavefront sensor.

A wavefront sensor is used as a direct observation tool to image the Gouy phase shift in photonic nanojets created by micrometer-sized dielectric spheres. The amplitude and phase distributions of light are found in good agreement with a rigorous electromagnetic computation. Interestingly the observed phase shift when travelling through the photonic jet is a combination of the awaited π Gouy shift and a phase shift induced by the bead refraction. Such direct spatial phase shift observation using wavefront sensors would find applications in microscopy, diffractive optics, optical trapping, and point spread function engineering.

Wide-field vibrational phase imaging.

We propose and implement a wide-field microscopy method to retrieve the real and imaginary part of a field emitted by coherent and resonant molecular scatterers. The technique is based on wave-front sensing and does not require the use of any reference beam. We exemplify its ability in wide-field coherent anti-Stokes Raman scattering imaging and retrieve the complex anti-Stokes field while spectrally scanning a molecular vibrational resonance. This approach gives access to the background-free Raman spectrum of the targeted molecular bond.

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.

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.

Large field-of-view phase and fluorescence mesoscope with microscopic resolution.

Phase and fluorescence are complementary contrasts that are commonly used in biology. However, the coupling of these two modalities is traditionally limited to high magnification and complex imaging systems. For statistical studies of biological populations, a large field-of-view is required. We describe a 30 mm2 field-of-view dual-modality mesoscope with a 4-µm resolution. The potential of the system to address biological questions is illustrated on white blood cell numeration in whole blood and multiwavelength imaging of the human osteosarcoma (U2-OS) cells.