Numerous biophysical questions require the quantification of short-range interactions between (functionalized) surfaces and synthetic or biological objects such as cells. Here, we present an original, custom built setup for reflection interference contrast microscopy that can assess distances between a substrate and a flowing object at high speed with nanometric accuracy. We demonstrate its use to decipher the complex biochemical and mechanical interplay regulating blood cell homing at the vessel wall in the microcirculation using an in vitro approach. We show that in the absence of specific biochemical interactions, flowing cells are repelled from the soft layer lining the vessel wall, contributing to red blood cell repulsion in vivo. In contrast, this so-called glycocalyx stabilizes rolling of cells under flow in the presence of a specific receptor naturally present on activated leucocytes and a number of cancer cell lines.
In bone tissue, multiscale interfaces provide the structural basis of essential bone functions and contribute to its macroscopic mechanical properties. The lacuno-canalicular network (LCN) hosting the osteocytes in the bone matrix, in particular, represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. We have demonstrated that label-free third-harmonic generation (THG) microscopy reveals the structure of the LCN in 3D with submicron precision over millimetric fields of view compatible with histology and can be coupled to second-harmonic generation (SHG) signals relating to the collagen organization in the bone matrix. Taking advantage of these label-free imaging methods, we investigate the impact of microgravity on the LCN structure in mice following a 1- month space flight. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.
Nonlinear optical microscopy is a biocompatible avenue for probing ordered molecular assemblies in biological tissues. As in linear optics, the nonlinear optical response from ordered systems is polarization-sensitive. This dependence can be used to identify and characterize local molecular ordering with micrometer-scale 3D resolution in a nonlinear microscope. In particular, third-harmonic generation (THG) microscopy is a nonlinear optical modality sensitive to the electronic nonlinear susceptibility χ(3) of a material. THG microscopy can be used to map χ(3) spatial variations (i.e. material interfaces), and to probe birefringence. In principle, polarization-resolved THG (P-THG) can therefore be used to probe ordered molecular arrays. However, the orientation, distribution, and nonlinear optical properties of the molecules near the beam focus all affect the detected signal. It is therefore necessary to develop a theoretical method which decouples these effects and permits the extraction of orientational information from P-THG images. In this report, we first present P-THG images of model systems (lipid droplets, multilamellar lipid vesicles) and biological tissues (human skin biopsy) which establish that P-THG is sensitive to lipid ordering and that it is maximized when excitation polarization is parallel to the ordered lipid molecules, giving impetus for the development of a thorough theoretical analysis. We then outline a multiscale model spanning the molecular (nm) and ensemble (μm) scales predicting the PTHG signal, consisting of three main steps: (i) calculation of the molecular electronic hyperpolarizability; (ii) determination of the anisotropic χ(3) for various molecular distribution parameters; and (iii) numerical calculations of the P-THG signal from lipid-water interfaces. This analysis links the measured P-THG response to lipid molecular structure and ordering.
Modal sensorless adaptive optics relies on the use of an image quality metric to estimate the amplitude of
aberrations, and of a well-suited set of aberration modes to describe the aberration. This set is chosen so that
aberration of one mode does not influence correction in another mode. In this paper, we show how these modes
can be derived experimentally, and investigate the influence of imperfect crosstalk removal on the accuracy of
correction. We show that the resulting error can be mitigated using appropriate algorithms that can incorporate
knowledge of the influence of the modes on the metric and, if available, partial knowledge of the aberrations.
Finally, we derive from these results the minimum time required for correction in various situations.
We investigate the parameters governing the accuracy of correction in modal sensorless adaptive optics for
microscopy. In this paper we focus on the case of two-photon excited fluorescence. Using analytical, numerical
and experimental results, we show that using a suitable number of measurements, accurate correction can
be achieved for up to 2 rad rms initial aberrations even without optimisation of the correction modes. We
demonstrate that this correction can be achieved using low light levels to minimise photobleaching and toxicity,
and we provide examples of such optimised correction.
Accurate control over the phase and amplitude modulation in an adaptive microscope is essential to the quality
of aberration correction that can be achieved. In this paper we present a robust and compact method for
characterising such amplitude and phase modulation in the pupil plane of the focussing objective. This method,
based on phase diversity, permits calibrating the microscope as a whole and thus avoids errors in the alignment
of the wavefront shaping device after calibration and the resulting imprecision in the induced modulation: by
acquiring three 2D images of the point spread function at different distances from the focal plane, we show that
the electric field distribution at the pupil plane can be retrieved using an iterative algorithm. We have applied
this technique to the characterisation of the phase modulation induced by a deformable mirror when conjugated
with the entrance pupil of different objectives, which permits accurate evaluation of the performance of the
mirror for subsequent aberration correction.
In adaptive microscope systems, it is often desirable to dispense with the wavefront sensor and perform aberration
correction through optimisation an appropriate quality metric, such as image brightness or sharpness. A sequence
of trial aberrations is applied to the adaptive element and the metric values are calculated. The optimum
aberration correction is derived from these measurements. An important choice in the design of these correction
schemes is the modal aberration expansion. This choice may depend upon several factors, such as the deformable
mirror, the optimisation metric, the aberration statistics or the image properties. We discuss these factors with
particular reference to microscope imaging.
Specimen-induced aberrations are frequently encountered in high resolution microscopy, particularly when high
numerical aperture lenses are used to image deep into biological specimens. These aberrations distort the focal
spot causing a reduction in resolution and, often more importantly, reduced signal level and contrast. This is
particularly problematic in multiphoton microscopy, where the
non-linear nature of the signal generation process
means that the signal level is strongly affected by changes in the focal spot intensity. We have developed an
adaptive two-photon fluorescence microscope to correct for these aberrations. Unlike a conventional adaptive
optics system, the microscope does not include a wavefront sensor but uses an efficient sensorless optimisation
scheme to obtain optimum aberration correction.
We use a vector field model to analyze the third-harmonic generation (THG) emission patterns for isolated objects
illuminated by a Gaussian beam. Simulations and experiments indicate that THG from biological (dielectric) structures is
essentially forward-directed, as opposed to e.g. THG from gold particles. We then address the issue of epidetecting
forward-emitted light backscattered in a turbid medium. We use Monte Carlo simulations and measurements to analyze
the effect of tissue properties (absorption, scattering), and of the geometry of the collecting optics. This analysis provides
guidelines for optimizing epidetection in coherent nonlinear microscopy.
An erratum is attached.
We present an overview of a wavefront sensor-less adaptive optics scheme for microscopy based upon the optimisation
of a metric related to the spatial frequency content of images. Aberrations are expanded as a series of
suitable functions that permit the independent optimisation of each aberration mode. A general scheme to derive
these modes theoretically and experimentally is presented. Resulting aberration correction is demonstrated in
an incoherent transmission imaging system and in a structured illumination microscope.
We present an overview of a wave front sensor-less adaptive optics scheme based upon the optimisation of the low spatial frequency content of images. Aberrations are expanded as a series of Lukosz functions as this permits the independent optimisation of each aberration mode. The scheme is demonstrated in an incoherent transmission microscope. It is shown how the method is related to so-called 'image sharpening' techniques.
We used intrinsic Second Harmonic Generation (SHG) by fibrillar collagen to visualize the three-dimensional
architecture of collagen fibrosis at the micrometer scale using laser scanning nonlinear microscopy. We showed that
SHG signals are highly specific to fibrillar collagen and provide a sensitive probe of the micrometer-scale structural
organization of collagen in tissues. Moreover, recording simultaneously other nonlinear optical signals in a multimodal
setup, we visualized the tissue morphology using Two-Photon Excited Fluorescence (2PEF) signals from endogenous
chromophores such as NADH or elastin. We then compared different methods to determine accurate indexes of collagen
fibrosis using nonlinear microscopy, given that most collagen fibrils are smaller than the microscope resolution and that
second harmonic generation is a coherent process. In order to define a robust method to process our three-dimensional
images, we either calculated the fraction of the images occupied by a significant SHG signal, or averaged SHG signal
intensities. We showed that these scores provide an estimation of the extension of renal and pulmonary fibrosis in
murine models, and that they clearly sort out the fibrotic mice.
Third-harmonic generation (THG) microscopy can provide structural information from unstained biological samples such as developing embryos. However, the contrast mechanisms in THG imaging need to be better characterized in order to develop practical applications. We studied experimentally and theoretically the influence of sample structure and excitation NA (Rayleigh length) on THG signals for various cases (spheres, interfaces). Because the third-harmonic signal critically depends on the spatial distribution of the Gouy shift, the effect of changing the excitation NA depends on the sample geometry within the focal volume. This phenomenon can be used to highlight certain structures within a complex system. Finally, we measured the nonlinear optical properties of several liquids, and we identified lipid bodies as an important source of contrast in biological THG imaging. We show that the technique can be used to characterize lipid accumulation in a variety of cells and tissues.
The regulation of morphogenetic movements that shape an embryo during its development remains a challenging issue in developmental biology, and may in certain cases involve mechanical sensitivity. Addressing this issue requires novel experimental approaches. We show that the combination of femtosecond laser pulse-induced ablation and multiphoton microcopy can be used to modulate and quantify morphogenetic movements in Drosophila embryos. We characterized the effects of focused nanoJoule pulse trains in developing embryos. We used targeted ablations to locally modify the embryo structural integrity and modulate morphogenesis. Femtosecond-pulse induced ablation was combined with nonlinear microscopy based on two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG).Correlation-based analysis of microscopy data allowed us to track the outcome of ablations and to analyze tissue deformations. These experiments provided insight into the interplay between gene expression and tissue deformations in developing embryos.
Animal embryo development exhibits a complex ensemble of cell movements that are tightly regulated by developmental gene expression. It was proposed recently that mechanical factors may also play an important role during development. Investigating these dynamical processes is technically challenging and requires novel in vivo investigation methods. We show that multiphoton microscopy can be used for both perturbing and analyzing morphogenetic movements in vivo. (i) nonlinear microscopy is well adapted for the sustained imaging of early Drosophila embryos despite their highly scattering nature; (ii) femtosecond pulse-induced ablation can be used to process specific tissues in vivo. Combining this approach with multimodal microscopy (two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG)), we report the successful quantitative modulation of morphogenetic movements in vivo. Our data provides insight to the issue of morphogenesis regulation.
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