scholarly journals Autofluorescence of stingray skeletal cartilage: hyperspectral imaging as a tool for histological characterization

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Júlia Chaumel ◽  
María Marsal ◽  
Adrián Gómez-Sánchez ◽  
Michael Blumer ◽  
Emilio J. Gualda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Sheet ◽  
Label Free ◽  
Cell Nuclei ◽  
Micro Computed Tomography ◽  
Composite Tissue ◽  
Light Sheet Microscopy ◽  
Imaging Tool ◽  
Biochemical Signals ◽  
First Time

AbstractTessellated cartilage is a distinctive composite tissue forming the bulk of the skeleton of cartilaginous fishes (e.g. sharks and rays), built from unmineralized cartilage covered at the surface by a thin layer of mineralized tiles called tesserae. The finescale structure and composition of elasmobranch tessellated cartilage has largely been investigated with electron microscopy, micro-computed tomography and histology, but many aspects of tissue structure and composition remain uncharacterized. In our study, we demonstrate that the tessellated cartilage of a stingray exhibits a strong and diverse autofluorescence, a native property of the tissue which can be harnessed as an effective label-free imaging technique. The autofluorescence signal was excited using a broad range of wavelengths in confocal and light sheet microscopy, comparing several sample preparations (fresh; demineralized and paraffin-embedded; non-demineralized and plastic-embedded) and imaging the tissue at different scales. Autofluorescence varied with sample preparation with the signal in both plastic- and paraffin-embedded samples strong enough to allow visualization of finescale (≥ 1 μm) cellular and matrix structures, such as cell nuclei and current and former mineralization fronts, identifiable by globular mineralized tissue. A defined pericellular matrix (PCM) surrounding chondrocytes was also discernible, described here for the first time in elasmobranchs. The presence of a PCM suggests similarities with mammalian cartilage regarding how chondrocytes interact with their environment, the PCM in mammals acting as a transducer for biomechanical and biochemical signals. A posterior analysis of hyperspectral images by an MCR-ALS unmixing algorithm allowed identification of several distinct fluorescence signatures associated to specific regions in the tissue. Some fluorescence signatures identified could be correlated with collagen type II, the most abundant structural molecule of cartilage. Other fluorescence signatures, however, remained unidentified, spotlighting tissue regions that deserve deeper characterization and suggesting the presence of molecules still unidentified in elasmobranch skeletal cartilage. Our results show that autofluorescence can be a powerful exploratory imaging tool for characterizing less-studied skeletal tissues, such as tessellated cartilage. The images obtained are largely comparable with more commonly used techniques, but without the need for complicated sample preparations or external staining reagents standard in histology and electron microscopy (TEM, SEM).

Exploring Morphological and Biochemical Linkages in Fungal Growth with Label-Free Light Sheet Microscopy and Raman Spectroscopy

ChemPhysChem ◽  
2016 ◽  
Vol 18 (1) ◽  
pp. 72-78 ◽  
Author(s):  
Soumik Siddhanta ◽  
Santosh Kumar Paidi ◽  
Kathryn Bushley ◽  
Ram Prasad ◽  
Ishan Barman
Keyword(s):  
Raman Spectroscopy ◽  
Fungal Growth ◽  
Light Sheet ◽  
Label Free ◽  
Light Sheet Microscopy

scholarly journals Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon I. R. Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Biological Samples ◽  
3D Imaging ◽  
Second Harmonic ◽  
Three Dimensional ◽  
Light Sheet ◽  
Optical Methods ◽  
Label Free ◽  
Light Sheet Microscopy

AbstractLight sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

Advances in imaging feto-placental vasculature: new tools to elucidate the early life origins of health and disease

2020 ◽  
pp. 1-11
Author(s):  
Yutthapong Tongpob ◽  
Caitlin Wyrwoll
Keyword(s):  
Ex Vivo ◽  
Light Sheet ◽  
Experimental Models ◽  
Cardiovascular Development ◽  
Micro Computed Tomography ◽  
Placental Function ◽  
Light Sheet Microscopy ◽  
Health And Disease ◽  
Insight Into

Abstract Optimal placental function is critical for fetal development, and therefore a crucial consideration for understanding the developmental origins of health and disease (DOHaD). The structure of the fetal side of the placental vasculature is an important determinant of fetal growth and cardiovascular development. There are several imaging modalities for assessing feto-placental structure including stereology, electron microscopy, confocal microscopy, micro-computed tomography, light-sheet microscopy, ultrasonography and magnetic resonance imaging. In this review, we present current methodologies for imaging feto-placental vasculature morphology ex vivo and in vivo in human and experimental models, their advantages and limitations and how these provide insight into placental function and fetal outcomes. These imaging approaches add important perspective to our understanding of placental biology and have potential to be new tools to elucidate a deeper understanding of DOHaD.

scholarly journals Integrative quantitative-phase and airy light-sheet imaging

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
N. R. Subedi ◽  
P. S. Jung ◽  
E. L. Bredeweg ◽  
S. Nemati ◽  
S. E. Baker ◽  
...  
Keyword(s):  
Evolutionary Biology ◽  
Imaging System ◽  
Light Sheet ◽  
Live Cells ◽  
Phase Imaging ◽  
Label Free ◽  
Quantitative Phase Imaging ◽  
Quantitative Phase ◽  
Light Sheet Microscopy ◽  
Order Of Magnitude

AbstractLight-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free recognition of cells and organelles, and quantifies their number-density that, thermodynamically, is more representative of metabolism than size. Here, we report the fusion of these two imaging modalities onto a standard inverted microscope that retains compatibility with microfluidics and open-source software for image acquisition and processing. An accelerating Airy-beam light-sheet critically enabled imaging areas that were greater by more than one order of magnitude than a Gaussian beam illumination and matched exactly those of quantitative-phase imaging. Using this integrative imaging system, we performed a demonstrative multivariate investigation of live-cells in microfluidics that unmasked that cellular noise can affect the compartmental localization of metabolic reactions. We detail the design, assembly, and performance of the integrative imaging system, and discuss potential applications in biotechnology and evolutionary biology.

scholarly journals Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dimitrios Kapsokalyvas ◽  
Rodrigo Rosas ◽  
Rob W. A. Janssen ◽  
Jo M. Vanoevelen ◽  
Miranda Nabben ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Light Sheet ◽  
Three Dimensions ◽  
Fluorescence Signal ◽  
Label Free ◽  
Microscopy Imaging ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy ◽  
3D Image Reconstruction

AbstractImaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy—imaging the sample from different angles followed by 3D image reconstruction—was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

scholarly journals BigDataProcessor2: A free and open-source Fiji plugin for inspection and processing of TB sized image data

2020 ◽  
Author(s):  
Christian Tischer ◽  
Ashis Ravindran ◽  
Sabine Reither ◽  
Rainer Pepperkok ◽  
Nils Norlin
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Imaging Techniques ◽  
Image Data ◽  
Light Sheet ◽  
Sensor Technology ◽  
Time Points ◽  
Light Sheet Microscopy ◽  
Link Type ◽  
Image Datasets

SummaryModern bioimaging and related areas such as sensor technology has seen tremendous development the last years allowing several contemporary imaging techniques, electron microscopy (EM) and light sheet microscopy in particular, to generate datasets frequently reaching the size of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2 – a Fiji plugin facilitating processing workflows for TB sized image datasets.Availability and implementationBigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2)[email protected], [email protected]

scholarly journals BigDataProcessor2: a free and open-source Fiji plugin for inspection and processing of TB sized image data

2021 ◽  
Author(s):  
Christian Tischer ◽  
Ashis Ravindran ◽  
Sabine Reither ◽  
Nicolas Chiaruttini ◽  
Rainer Pepperkok ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Imaging Techniques ◽  
Image Data ◽  
Light Sheet ◽  
Supplementary Information ◽  
Sensor Technology ◽  
Supplementary Data ◽  
Time Points ◽  
Light Sheet Microscopy

Abstract Summary Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2—a Fiji plugin facilitating processing workflows for TB sized image datasets. Availability and implementation BigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2). Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals CloudReg: Automatic Terabyte-Scale Cross-Modal Brain Volume Registration

2021 ◽  
Author(s):  
Vikram Chandrashekhar ◽  
Daniel J Tward ◽  
Devin Crowley ◽  
Ailey K Crow ◽  
Matthew A Wright ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Brain Magnetic Resonance Imaging ◽  
Light Sheet ◽  
Imaging Data ◽  
Micro Computed Tomography ◽  
Light Sheet Microscopy ◽  
Volume Registration ◽  
Scalable Analysis ◽  
Spatially Varying

AbstractQuantifying terabyte-scale multi-modal human and animal imaging data requires scalable analysis tools. We developed CloudReg, an open-source, automatic, terabyte-scale, cloud-based image analysis pipeline that pre-processes and registers cross-modal volumetric datasets with artifacts via spatially-varying polynomial intensity transform. CloudReg accurately registers the following datasets to their respective atlases: in vivo human and ex vivo macaque brain magnetic resonance imaging, ex vivo mouse brain micro-computed tomography, and cleared murine brain light-sheet microscopy.

Second Harmonic Generation Light Sheet Microscopy (SHG-LSM): A new tool for label-free 3D bioimaging

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher J. Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Second Harmonic ◽  
Light Sheet ◽  
Label Free ◽  
Light Sheet Microscopy

scholarly journals Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Manuel Alonso Y Adell ◽  
Simona M Migliano ◽  
Srigokul Upadhyayula ◽  
Yury S Bykov ◽  
Simon Sprenger ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Atpase Activity ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Escrt Machinery ◽  
Steady Growth ◽  
Bud Neck ◽  
Membrane Scission ◽  
Membrane Budding ◽  
Quantitative Fluorescence

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

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