In this contribution, we aim to compare the accuracy of a large variety of quantitative phase imaging (QPI) techniques (phase shifting, off axis, etc) in terms of trueness and precision. For this purpose, we developed a numerical toolbox capable of computing the electromagnetic field at the sample plane of arbitrarily complex microscopes, in the presence of any microscale object at the sample plane. The discussions are based on the numerical imaging of nanoparticles, micro-beads, bacteria, 2D materials and mammalian cells. This way, we could discuss the benefits and limitations of all the existing QPI techniques to help better chose the right tool as a function of the application.
We demonstrate a novel approach to super-resolution in stimulated Raman scattering based on single-pixel detection scheme. We exploit speckle structured illumination, and enable fast super-resolution microscopy using Raman contrasts. The methodology is straightforward to implement and provides label-free super-resolution imaging of optically thick specimens, therefore paving the way for probing complex biological systems when exogenous labelling is challenging.
We revisit wide field CARS imaging using speckle field illuminations. In the proposed scheme we break the CARS coherence using fast varying pump speckle illuminations while keeping static the Stokes speckle. Acquiring a large number of Stokes images enables dynamic speckle illumination (DSI) and random illumination microscopy (RIM). We show that the first one enables quasi-confocal axial sectioning (1/z) while the second one, through post-processing, can retrieve a superior image contrast, noise level and spatial resolution as an important step towards robust nonlinear super-resolution CARS microscopy.
Total Internal Reflection Fluorescence Microscopy (TIRFM) exploits an evanescent field induced at the boundary between high and low refractive index media to selectively excite the sample inside a very thin region (from 100 to 300 nm depending on the illumination angle) above the coverslip surface. The minimum exposure of the sample to light above the excitation slice reduces significantly the out-of-focus fluorescence and phototoxicity which are major issues in live-cell imaging. It has become an indispensable tool in biology, in particular to study the molecular traffic at the cell plasma membranes.
However, in many applications, the lateral resolution of TIRF, which is diffraction limited to about 300 nm, is not sufficient. In addition, the optical sectioning of the evanescent illumination of TIRF is seldom perfect. Propagative waves stemming from imperfections in the optical train of the instrument and/or light scattering by the sample itself are able to excite the fluorescence in the volume of the sample. When the latter is densely marked, these leaks result in out-of-focus fluorescence which deteriorates the signal to noise ratio.
To improve simultaneously the lateral resolution and the image contrast, and to address the difficulties related to the control of the illumination patterns, we propose to adapt the recently developed Random Illumination Microscope (RIM) to the TIR configuration. We show that this approach yields a two-fold resolution gain and ameliorates the image contrast without compromising the ease of use of standard TIRFM. We apply TIRF-RIM to calibrated targets and to fixed and live biological samples with a sub-100nm resolution.
Raman microscopy is a powerful mode of label free nonlinear optical microscopy that is hampered by weak cross-sections, leading to slow imaging. We will discuss two advances in coherent Raman microscopy: 1) high speed coherent anti-Stokes Raman scattering microscopy imaging based on spatial frequency modulation imagining (SPIFI) where a structured line focus is used to image is used to image specimens with a single pixel detector. 2) Doppler Raman microscopy that exploits the extremely low timing jitter of modelocked lasers for ultrasensitive Raman spectroscopy and microscopy.
Raman microscopy has been widely developed for label free nonlinear optical microscopy of biological systems. Imaging speed in these systems is hampered by low Raman scattering cross sections and the requirement of scanning a focused laser beam through the sample in conventional Raman microscopy. The serial acquisition that is necessary in point scanning microscopy slows image acquisition and limits the dwell time are each image pixel. Here, we discuss two new imaging methods that are based on spatial frequency modulation imagining (SPIFI) [1-2], where a structured line focus is used to image is used to image specimens by collecting light on a single pixel detector. We discuss the use of SPIFI to improve the imaging speed of Spontaneous Raman scattering and coherent anti-Stokes Raman scattering microscopy. A detailed noise analysis highlighting the advantages and disadvantages of SPIFI as compared to conventional point scan imaging is presented.
Conventional CARS microscopy requires scanning a point focus through the specimen limits imaging speed. We present a spatial frequency projection imaging (SPIFI) method for CARS microscopy to spatially multiplex CARS microscopy. A spinning disk modulator is used to rapidly modulate the Stokes field with a rapidly swept spatially periodic transmission grating. SPIFI-CARS images are obtained by Fourier transforming the single pixel signal. Images of CARS and second harmonic generation from histological slices will be presented. The physics of image formation and the impact of multiplexing on SNR will be discussed. Prospects for scaling to high speed CARS imaging will be discussed.
We present a simulation of stimulated Raman scattering that could make deep super resolution imaging possible with chemical selectivity and relatively low power levels. For this, we directly use speckles as a structured illumination pattern in a coherent Raman scattering processes, in particular Stimulated Raman Scattering. Using off-the-shelf conjugate gradient-based algorithm, we demonstrate that the method enables super-resolution better than conventional raster scanning techniques.
Quantitative phase imaging (QPI) has become an important imaging modality providing rapid, label-free measurements of a biological structure’s morphology and permittivity. In particular, reflection QPI systems are advantageous for their improved sensitivity to high-resolution axial structures and their ability to image thin and thick tissues alike. Existing reflection modalities often utilize interferometric setups requiring specialized system designs that limit their application in widespread biological research. We developed reflection intensity diffraction phase microscopy (rIDPM) to provide an easily accessible reflection QPI system for biological imaging applications. This new modality recovers a biological structure’s phase from intensity-only measurements using a standard reflection microscope modified with a translatable light source. We derived inverse scattering models for rIDPM addressing the common imaging condition of biological cells on a glass sample slide. Our models utilize the first Born approximation with a semi-infinite, partially reflective boundary condition accounting for reflections from the glass slide interface. The resulting volumetric model is linear and easily implementable providing fast, computationally efficient recovery of the object’s complex permittivity. Under these imaging conditions, we show forward-scattered fields primarily contribute to the final intensity image for objects taller than half the illumination wavelength. We show this rIDPM modality provides improved contrast of subcellular features from unstained HeLa cell samples compared to existing QPI transmission systems. We also demonstrate our model’s flexibility in recovering high-frequency features with improved contrast from designed annular illumination patterns.
In deep photoacoustic imaging, resolution is inherently limited by acoustic diffraction, and ultrasonic frequencies cannot be arbitrarily increased because of attenuation in tissue. Here we report on the use of multiple speckle illumination to perform super resolution photoacoustic imaging. We show that the analysis of speckle-induced second-order fluctuations of the photoacoustic signal combined with deconvolution enables to resolve optically absorbing structures below the acoustic diffraction limit.
We explain how to design a high Q, polarization independent, angularly tolerant filter, with a "doubly
periodic" resonant grating etched in a high index material. Thanks to its broad angular acceptance, the
fabricated component shows a Q factor of 5600 with a 580μm diameter Gaussian beam. Performing a detailed
theoretical and experimental comparative study, we identify the parameters responsible for the degradation
of the performances of the filter.
A resonant grating filter can be basically described as a sub-wavelength grating inscribed on a a planar
waveguide made of dielectric thin film layers. The reflectivity of such a structure presents some peaks versus the
wavelength that are generated by the coupling and coupling out of different modes of the waveguide. These
peaks can be tailored in order to create free-space narrow bandpass inverse (notch) filters, with a very sharp
spectral response (typically a spectral bandwidth below 0.5 reflectance nm for a component working at 1550nm).
We show an experimental demonstration of a high performances resonant grating filter working in reflectance
under a given incidence and wavelength. This filter presents simultaneously a good angular tolerance and
polarization independence with a full width at half maximum about 0.4nm. The dielectric stack reflectance
configuration is chosen so that the coupling conditions for two TE reflectance guided modes are simultaneously
satisfied. Moreover, the grating cell presents a complex structure of four holes with different diameters,
optimized for increasing the angular tolerance of the filter while maintaining a narrow spectral width for any
polarization. Comparisons with theoretical calculations are provided.
We developed an approximate method to express the effects induced by a periodic perturbation on the dispersion relation of the eigenmodes of a planar waveguide. In the particular case when the grating has a plane of symmetry, we show that the dispersion relation of the eigenmodes can present a point of degeneracy. We relate the existence of a point of degeneracy to the possibility to obtain resonances, at the same wavelength and at the same angle of incidence, for both s and p polarization. This property is important for designing nonpolarizing guided mode resonant filters for oblique incidence.
We propose a technique, based on an interferometric experiment and an optimization algorithm, that permits to retrieve the map of permittivity of unknown objects deposited on a flat surface. We show numerically that one can obtain a better resolution than the usual Rayleigh limit even with detectors and emitters in the far-field zone.
For many applications, reducing the total amount of the light scattered from rough surfaces is a crucial problem. We show that a scattering-reduction effect is obtained by coating rough absorbing surfaces. Numerical results as well as experimental ones are presented. We also study the case of inhomogeneous structures with embedded scatterers.
The basic principles of guided mode resonance filters constituted by a high index modulated coating deposited on a classical glass substrate are presented. A rigorous method used to compute the reflection coefficients of this kind of structures is described. We show that in some cases, computation can be made by using an approximate method. These structures, which permit one to design narrowband inverse filters, are compared to solutions based on classical multidielectric coatings.
Calculation of the laser light diffraction by shallow (with respect to the wavelength) harmonically profiled reflecting surface of the dielectric is presented"
We consider the scattering of light by a system of randomly distributed particles above an interface. It is shown that this system can produce backscattering enhancement even for a very dilute system such that multiple scattering is negligible. A physical mechanisms is proposed and the role of the Fresnel reflectivity of the interface is examined. Differences between the behavior of s and p-polarization are considered.
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