Our study introduces a new method for label-free super-resolved polarimetry on nanomaterials, compatible with in-situ analysis. Integrating Image Scanning Microscopy (ISM) with polarimetry techniques, we achieve remarkable resolutions down to 90 nm while acquiring polarization information. Overcoming limitations associated with fluorophores in challenging materials, our approach facilitates quantitative measurements of optical properties. Applied successfully to nanostructured surfaces created by femtosecond lasers and boron nitride nanotubes, our work showcases the versatility of this methodology.
We propose a novel design to perform quantitative phase confocal imaging with super-resolution imaging capabilities. Our approach, derived from image scanning microscopy, allows to perform quantitative label-free images up to 24kHz for 2D imaging. We demonstrate the capability to quantitatively image living adherent cells and biological tissue slices.
Laser processing of material surfaces has been very known for the last five decades. Femtosecond LIPSS, are created generally on the surface, they could be classified into two groups depending on the periodicity of the structures: LSFL showing a periodicity lower than the incident wavelength (λ_l), and HSFL with a periodicity well below λ_l that could sit below the optical diffraction limit. In this paper, we show an unprecedented resolution of a noninvasive label-free optical method to observe such structures, that does not require a priori knowledge of the surface. We demonstrate that using a modified reflectance confocal microscope reflection (CMR), the characterization of HSFL(̴Λ_HSFL∽120 nm @ λ_l=257 nm) is possible and efficient. These results, pave the way toward a new, better, and more resolved optical technique to observe nanostructures below the diffraction limit.
Fluorescence microscopy has succeeded in attaining super-resolution localization of single emitters in cellular biology. However, 3D localization deep inside tissue is still challenging. A few years ago, we developed SELFI: self-interference 3D super-resolution microscopy, a framework for 3D single-molecule localization within multicellular specimens and tissues. Here, we extend the capability of SELFI to the near-infrared (NIR) region where carbon nanotubes (CNTs) are strong emitters. The aim of this work is to develop NIR SELFI for single-particle tracking applications of CNTs in live brain tissues or NIR quantum dots. SELFI uses a diffraction grating placed on the optical path of the sample image, generating an interference pattern within diffraction limited images of point emitters. A single image obtained with NIR SELFI contains two independent variables: the intensity distribution to extract the intensity centroid to determine the lateral localization, and the wavefront curvature (provided by the interfringes) to get the axial super-localization. SELFI was first developed to localize red emitting dyes and quantum dots. The performance of the system is examined by means of the standard deviation and root mean square error of the localizations. The experiments performed show that the 3D-precision and accuracy achieved with NIR SELFI are both below 100 nm for emission around 1000 nm and high photon budget. Therefore, we can now achieve 3D localization in the NIR, permitting 3D single-particle tracking of CNTs at video rate in complex environments.
In localization microscopy, the position of isolated fluorescent emitters are estimated with a resolution better than the diffraction limit. In order to image thick samples, which are common in biological applications, there is considerable interest in extending the depth-of-field of such microscopes in order to make their accuracy as invariant as possible to defocus. For that purpose, we propose to optimize annular binary phase masks placed in the pupil of the microscope in order to generate a point spread function for which the localization accuracy is almost invariant along the optical axis. The optimization criterion is defined as the localization accuracy in the plane expressed in terms of the Cram´er-Rao bound. We show that the optimal masks significantly increase the depth-of-field of single-molecule imaging techniques relatively to an usual microscope objective.
We describe a technique based on the use of a high-resolution quadri-wave lateral shearing interferometer to perform quantitative linear birefringence measurements on biological samples [1] such as living cells and tissues. The system combines QPI with different excitation polarizations to create retardance images. This creates a new kind of image contrast based on the local retardance, reveals the structure of sample anisotropic components and adds specificity to label-free phase images. We implemented this technique allowing us to take retardance images in less than 1 second which allows us to make high speed acquisitions to reconstruct tissues virtual slides with different modalities (i.e intensity, phase and retardance). Comparisons between healthy and tumoral 10 µm thick skin tissues and collagen orientation studies in the latter will be presented.
[1] S. Aknoun, P. Bon, J. Savatier, B. Wattellier, and S. Monneret, "Quantitative retardance imaging of biological samples using quadriwave lateral shearing interferometry," Opt. Express 23, 16383-16406 (2015).
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.
We describe the use of polarized spatially coherent illumination to perform linear retardance imaging and measurements of semi-transparent biological samples using a quantitative phase imaging technique [1].
Quantitative phase imaging techniques [2-5] are used in microscopy for the imaging of semi-transparent samples and gives information about the optical path difference (OPD). The strength of those techniques is their non-invasive (the sample is not labelled) and fast approach. However, this high contrast is non-specific and cannot be linked to specific properties of the sample.
To overcome this limitation, we propose to use polarized light in combination with QPI. Indeed, anisotropy has been used to reveal ordered fibrous structures in biological samples without any staining or labelling with polarized light microscopy [6-8]. Recent studies have shown polarimetry as a potential diagnostic tool for various dermatological diseases on thick tissue samples [9]. Particularly, specific collagen fibers spatial distribution has been demonstrated to be a signature for the optical diagnosis and prognosis of cancer in tissues [10].
In this paper, we describe a technical improvement of our technique based on high-resolution quadri-wave lateral shearing interferometry (QWLSI) and liquid crystal retarder to perform quantitative linear birefringence measurements on biological samples. The system combines a set of quantitative phase images with different excitation polarizations to create birefringence images. These give information about the local retardance and orientation of biological anisotropic components.
We propose using a commercial QWLSI [11] (SID4Bio, Phasics SA, Saint Aubin, France) directly plugged onto a lateral video port of an inverted microscope (TE2000-U, Nikon, Japan). We are able to take retardance images in less than 1 second which allows us to record dynamic phenomena (living cells study) and make high speed acquisitions to reconstruct tissues virtual slides. Results on living cells and comparisons between healthy and tumoral 10 µm thick skin tissues will be presented.
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.
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.
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.
We present a new technique based on the self-interference of Supercritical Angle Fluorescence (SAF) emission in
order to perform full-field cell membrane imaging. We show that our Point Spread Function (PSF) engineering
technique allows us to obtain a 100 nm axial sectioning while conserving the original lateral resolution of the
microscope. The images are acquired using an optical module that can be connected to any fluorescent microscope
to simultaneously monitor in real time both the cell membrane and in-depth phenomena.
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.
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.
In this paper, we discuss the possibility of making a super-axially-resolved image of a biological sample using
supercritical angle diffusion. This labeling-free approach is suitable to any microscope equipped with a NAobj <
1.33 microscope objective and can be used either for conventional intensity imaging or for quantitative phase
imaging. We expose some results on beads an cells showing the potential of this method.
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.
Phase imaging with a high-resolution wavefront sensor is a useful setup for biological imaging. Our setup is based
on a quadriwave lateral shearing interferometer mounted on a commercial non-modified transmission white-light
microscope. That allows us to make simultaneous measurement in both quantitative transmission phase and
fluorescence imaging. We propose here to study co-localization between phase and fluorescence on african
green monkey kidney COS7 cells. Phase permits an enhanced visualization of the whole cell and intracellular
components while the fluorescence allows a complete identification of each component. Post treatments on phase-shift
images are proposed and become very interesting for enhanced visualization of small details such as vesicles
or mitochondrias.
Phase imaging with a high-resolution wavefront sensor is a useful setup for biological imaging. Our setup is based
on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope.
We propose here to study long-time duration imaging on different type of adherent cells: green monkey kidney
COS7 cells, human breast epithelial MCF10A cells, and human breast cancer derived from MDA-231 cells. This
study permits a enhanced visualization of the whole cell life at different levels of confluence. Post treatments
on phase-shift images are proposed and become very interesting for enhanced visualization of small details and thresholding.
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