Structured Illumination Raman Microscopy for High-Resolution Label-Free Imaging

The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.

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Label-Free Live-Cell Imaging with Confocal Raman Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2011.12.027 ◽

2012 ◽

Vol 102 (2) ◽

pp. 360-368 ◽

Cited By ~ 117

Author(s):

Katharina Klein ◽

Alexander M. Gigler ◽

Thomas Aschenbrenner ◽

Roberto Monetti ◽

Wolfram Bunk ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Raman Microscopy ◽

Live Cell ◽

Confocal Raman Microscopy ◽

Label Free ◽

Confocal Raman

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Optical Imaging of the Small Intestine Immune Compartments Across Scales

10.21203/rs.3.rs-1046138/v1 ◽

2021 ◽

Author(s):

Arielle Planchette ◽

Cédric Schmidt ◽

Olivier Burri ◽

Mercedes Gomez de Agüero ◽

Aleksandra Radenovic ◽

...

Keyword(s):

Spatial Distribution ◽

Small Intestine ◽

High Resolution ◽

Imaging Modality ◽

Regions Of Interest ◽

Label Free ◽

3 Dimensional ◽

Wide Range ◽

Mouse Small Intestine ◽

Made In

Abstract The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues, with label-free capacities for the observation of auto-fluorescent signals as well fluorescent-labelled targets of interest in multiple channels. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify density of villi throughout centimetre long segments of intestine. Furthermore, we exhibit the age- and microbiota-dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D image acquisition with the reverse-OPT sample preparation and a 2D high-resolution imaging modality adds value to interpretations made in 3D. This cross-modality referencing technique is found to provide accurate localisation of ROIs and to add value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment.

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Super-Resolution Label-free Volumetric Vibrational Imaging

10.1101/2021.01.08.425961 ◽

2021 ◽

Author(s):

Chenxi Qian ◽

Kun Miao ◽

Li-En Lin ◽

Xinhong Chen ◽

Jiajun Du ◽

...

Keyword(s):

High Resolution ◽

Three Dimensional ◽

Super Resolution ◽

Label Free ◽

Fluorescence Techniques ◽

Biological Structures ◽

Imaging Strategy ◽

Endogenous Proteins ◽

Low Efficiency ◽

Protein Rich

Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. However, super-resolution fluorescence techniques, including both optics-oriented and sample-expansion based, are limited in quantification and throughput especially in tissues from photobleaching or quenching of the fluorophores, and low-efficiency or non-uniform delivery of the probes. Here, we report a general sample-expansion vibrational imaging strategy, termed VISTA, for scalable label-free high-resolution interrogations of protein-rich biological structures with resolution down to 82 nm. VISTA achieves decent three-dimensional image quality through optimal retention of endogenous proteins, isotropic sample expansion, and deprivation of scattering lipids. Free from probe-labeling associated issues, VISTA offers unbiased and high-throughput tissue investigations. With correlative VISTA and immunofluorescence, we further validated the imaging specificity of VISTA and trained an image-segmentation model for label-free multi-component and volumetric prediction of nucleus, blood vessels, neuronal cells and dendrites in complex mouse brain tissues. VISTA could hence open new avenues for versatile biomedical studies.

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A technique for resolution assessment in blind-SIM experiments

10.1101/2021.09.01.458550 ◽

2021 ◽

Author(s):

Imen Boujmil ◽

Giancarlo Ruocco ◽

Marco Leonetti

Keyword(s):

High Resolution ◽

Biological Sample ◽

A Priori ◽

Resolution Enhancement ◽

Super Resolution ◽

Experimental Setup ◽

Wide Field ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Resolution Measurement

Super resolution techniques are an excellent alternative to wide field microscopy, providing high resolution also in (typically fragile) biological sample. Among the various super resolution techniques, Structured Illumination Microscopy (SIM) improve resolution by employing multiple illumination patterns to be deconvolved with a dedicated software. In the case of blind SIM techniques, unknown patterns, such as speckles, are used, thus providing super resolved images, nearly unaffected by aberrations with a simplified experimental setup. Scattering Assisted Imaging, a special blind SIM technique, exploits an illumination PSF (speckle grains size), smaller than the collection PSF (defined by the collection objectives), to surpass the typical SIM resolution enhancement. However, if SAI is used, it is very difficult to extract the resolution enhancement form a priori considerations. In this paper we propose a protocol and experimental setup for the resolution measurement, demonstrating the resolution enhancement for different collection PSF values.

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