Label-free multimodal nonlinear optical microscopy reveals features of bone composition in pathophysiological conditions.

Bone tissue features a complex microarchitecture and biomolecular composition, which determine biomechanical properties. In addition to state-of-the-art technologies, innovative optical approaches allowing the characterization of the bone in native, label-free conditions can provide new, multi-level insight into this inherently challenging tissue. Here, we exploited multimodal nonlinear optical (NLO) microscopy, including co-registered stimulated Raman scattering, two-photon excited fluorescence, and second-harmonic generation, to image entire vertebrae of murine spine sections. The quantitative nature of these nonlinear interactions allowed us to extract accurate biochemical, morphological, and topological information on the bone tissue and to highlight differences between normal and pathologic samples. Indeed, in a murine model showing bone loss, we observed increased collagen and lipid content as compared to the wild type, along with a decreased craniocaudal alignment of bone collagen fibres. We propose that NLO microscopy can be implemented in standard histopathological analysis of bone in preclinical studies, with the ambitious future perspective to introduce this technique in the clinical practice for the analysis of larger tissue sections.

Fingerprint multiplex CARS at high speed based on supercontinuum generation in bulk media and deep learning spectral denoising.

We introduce a broadband coherent anti-Stokes Raman scattering (CARS) microscope based on a 2-MHz repetition rate ytterbium laser generating 1035-nm high-energy (≈µJ level) femtosecond pulses. These features of the driving laser allow producing broadband red-shifted Stokes pulses, covering the whole fingerprint region (400-1800 cm-1), employing supercontinuum generation in a bulk crystal. Our system reaches state-of-the-art acquisition speed (<1 ms/pixel) and unprecedented sensitivity of ≈14.1 mmol/L when detecting dimethyl sulfoxide in water. To further improve the performance of the system and to enhance the signal-to-noise ratio of the CARS spectra, we designed a convolutional neural network for spectral denoising, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues.

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

Composite Peptide-Agarose Hydrogels for Robust and High-Sensitivity 3D Immunoassays.

Canonical immunoassays rely on highly sensitive and specific capturing of circulating biomarkers by interacting biomolecular baits. In this frame, bioprobe immobilization in spatially discrete three-dimensional (3D) spots onto analytical surfaces by hydrogel encapsulation was shown to provide relevant advantages over conventional two-dimensional (2D) platforms. Yet, the broad application of 3D systems is still hampered by hurdles in matching their straightforward fabrication with optimal functional properties. Herein, we report on a composite hydrogel obtained by combining a self-assembling peptide (namely, Q3 peptide) with low-temperature gelling agarose that is proved to have simple and robust application in the fabrication of microdroplet arrays, overcoming hurdles and limitations commonly associated with 3D hydrogel assays. We demonstrate the real-case scenario feasibility of our 3D system in the profiling of Covid-19 patients' serum IgG immunoreactivity, which showed remarkably improved signal-to-noise ratio over canonical assays in the 2D format and exquisite specificity. Overall, the new two-component hydrogel widens the perspectives of hydrogel-based arrays and represents a step forward towards their routine use in analytical practices.

Experimental determination of shift-less aberration bases for sensorless adaptive optics in nonlinear microscopy.

Adaptive optics can improve the performance of optical systems and devices by correcting phase aberrations. While in most applications wavefront sensing is employed to drive the adaptive optics correction, some microscopy methods may require sensorless optimization of the wavefront. In these cases, the correction is performed by describing the aberration as a linear combination of a base of influence functions, optimizing an image quality metric as a function of the coefficients. The influence functions base is generally chosen to either efficiently represent the adaptive device used or to describe generic wavefronts in an orthogonal fashion. A rarely discussed problem is that most correction bases have elements which introduce, together with a correction of the aberration, a shift of the imaging field of view in three dimensions. While simple methods to solve the problem are available for linear microscopy methods, nonlinear microscopy techniques such as multiphoton or second harmonic generation microscopy require non-trivial base determination. In this paper, we discuss the problem, and we present a method for calibrating a shift-less base on a spatial light modulator for two-photon microscopy.

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.