They are thus hazardous and there is need for decontamination and inactivation procedures for laboratory surfaces and non-disposable material. We assessed the effectiveness of different reagents to clean and disassemble potentially pathogenic assemblies adsorbed on non-disposable materials in laboratories. We provide an integrated representation where desorption and neutralization efficacy and surface compatibility are combined to facilitate the choice of the most adapted decontamination procedure. This representation, together with good laboratory practices, contributes to reducing potential health hazards associated to manipulating protein assemblies with prion-like properties. Consequently, to minimize the risk associated to exposure of workers in the laboratory to those assemblies it is not only important to develop guidelines for how to best handle those assemblies in the laboratory but also to develop cleaning methods that allow reducing the potential hazard associated to their manipulation. Decontamination and inactivation procedures allowing health hazard reduction by a factor to have been implemented in the prion field for reusable material.
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Multicellular systems Abstract In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution.
The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium.
In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon FEP foils. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible.
Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters.
Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation.
Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days.
The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e. Download PDF Introduction Light Sheet Fluorescence Microscopy LSFM has revolutionized the imaging of three-dimensional specimens ranging from micrometres spheroids, organoids to centimetres biopsies, model organisms in diameter 1 , 2 , 3 , 4.
Light spots and light sheets based on conventional light sources have been used for more than one century. However, lasers are required for the true optical sectioning capability. While laser-based light sheet—based macroscopes had been built several times 6 , the performance of light sheet at a microscopic level was not known until Huisken et al. In , SPIM was applied for the imaging of 3D cell cultures in collagen gel and of multicellular tumor spheroids 8. The same year, Dodt et al.
The introduction of the Digitally-scanned Light Sheet Microscope DSLM replaced the static light sheet generated with a cylindrical lens with a scanned light sheet, resulting in a more flexible set-up with increased penetration depth and less illumination artefacts A further milestone, the invention of lattice light sheet microscope, opened LSFM to super-resolution capabilities Currently, a large diversity of LSFM setups exist for application in cell and developmental biology, pathology, neuroscience, and biophysics 13 , 14 , Ever since the introduction of LSFM, agarose and other hydrogels have been used as transparent and biocompatible mounting media to immobilise specimens during imaging 2 , 16 , While agarose-gel embedding is convenient for mounting drosophila and zebrafish embryos 7 , 11 , it is not suitable for most 3D cell cultures and generally inapplicable for mounting optically cleared specimens.
In 3D-cultures such as organoids, cells grow in soft hydrogels, e. Matrigel 18 , which are composed of extracellular matrix ECM proteins such as collagen and laminin Embedding 3D-cultures into agarose gels deforms both the ECM hydrogels as well as the delicate multi-cellular structures growing inside. Attempts have been made to avoid specimen embedding, e. Most importantly, soluble components rapidly diffuse in and out of the agarose gel, implying that the specimens cannot be properly isolated from potential contaminants of chemical or bacterial origin floating in the LSFM sample chamber In order to reduce strong light scattering effects caused by refractive index mismatches in three-dimensional dense tissues, optical clearing solutions are used to homogenise the refractive indices across the whole sample.
The impulse given by LSFM led to the ongoing development of new clearing protocols in the last decade. Dodt et al. Currently, two major families of optical clearing solutions are used: 1 organic solvents-based and 2 water-based clearing solutions 24 , Solvent-based optical clearing methods are e. Many water-based clearing protocols have been developed since These methods can be distinguished in 1 hydrogel embedding-based, such as CLARITY 28 , 2 immersion-based for which the refractive index matching is achieved just by immersion , e.
SeeDB 29 , and 3 hyperhydration-based, such as e. All these clearing protocols have drawbacks, and have been optimized for specific organs. Comparative studies assessed the applicability range of several methods 31 , Organic solvents for optical clearing are often chemically aggressive and damage the objective lenses or the LSFM sample chamber.
Thus, a common approach uses large glass or quartz cuvettes to contain the clearing medium, in which the specimen is immersed For this imaging approach, low magnification air objective lenses or macro lenses with low numerical apertures NAs are frequently used 33 , 34 , which tend to yield relatively low-resolution images.
Water-based clearing solutions are less corrosive compared to organic solvents and can be combined with immersion objective lenses with higher NAs, which allow for a higher resolution and a better image quality. Nevertheless, the handling of large volumes of optical clearing solutions can be challenging, e. In a previous publication, we used thin square cross section glass capillaries with an inner side length of about 1 mm for mounting small organically cleared specimens.
This allowed us to use immersion objective lenses in combination with less or non-corrosive media to fill the LSFM chamber Although we successfully applied this method to the in toto study of drug-treated spheroids 36 , connecting and sealing the capillary to the holder proved to be time-consuming.
The FEP-cylinders allowed the observation of 3D microtubule dynamics unhindered by rigid and flat surfaces The cuvettes are closed at the bottom end, which greatly simplifies handling compared to the previously used glass capillaries and FEP-cylinders. They allow fast and straightforward specimen mounting using a forceps or pipettes to deposit the specimen into the cuvettes, and facilitate full containment of the internal mounting medium. The ultra-thin FEP-foil cuvettes are compatible with aqueous media as well as with water-based and organic clearing solutions, which all differ in their refractive indices.
Our data show that ultra-thin FEP-foil cuvettes allow high quality imaging of native non-cleared and optically cleared 3D specimens, such as liver and pancreas organoids, thick murine kidney and brain tissue sections as well as whole murine ovaries. In order to demonstrate the suitability of the cuvette to handle both solvent-based and water-based clearing solutions, we used Ethanol-ECi and CUBIC2 solution, respectively.
Their usage is simple of use and they effectively clear our specimens. The image quality allows for the application of multiple-views image reconstruction and for the subsequent application of automated quantitative image analysis pipelines 42 , FEP-films have outstanding properties for light microscopy.
FEP-films are transparent with a refractive index of 1. Like most fluorocarbon plastics, FEP is chemically inert and resistant to organic solvents, acids and bases. The 3D drawings were exported to the stereolithographic file format. The positive moulds were produced by Shapeways Eindhoven, NL, www. Two positive moulds, with arrays of square and octagonal cross section pillars, respectively, were 3D-printed Fig. The acrylic polymer has the required mechanical and thermal properties to withstand the vacuum forming process.
Prior to their usage, the positive moulds are cleaned by immersion in an ultrasonic bath and inspected with a stereomicroscope. Smaller samples are deposited by pipetting right. The individual cuvettes are cut from the array with a scalpel.
Finally, individual cuvettes are glued onto stainless steel pins diameter 3 mm, length 20 mm using instant glue Pattex, Repair Extrem Fig. Mice were kept under 12 hours light and dark cycle with food and water ad libitum. Mouse perfusion was performed according to Stefani et al.
Prior to clearing, the brains were washed three times with PBS for 15 minutes, and the hemispheres were separated. Before imaging, the CUBIC2 solution was refreshed, and, using forceps, the blocks were inserted into octagonal ultra-thin FEP-foil cuvettes with a diameter of 5 mm Fig.
Prior to clearing, the kidney was washed three times with PBS for 15 minutes and cut in small blocks. Murine ovary: Explanted and ex vivo cultured ovaries, expressing GFP-cKit specifically in all oocytes, were treated as previously published 46 , Staining was performed as published by Smyrek et al. The ovaries were permeabilised with 0. Next, the ovaries were treated with blocking buffer 0. To avoid floating ovaries, it is important to make sure that no air bubbles enter the cuvette.
Figure 2 Specimens with heterogeneous optical densities mounted into ultra-thin FEP-foil cuvettes provide a high resolution at the subcellular level. Renderings, single central planes and detail views of: a A whole murine ovary of an eight day-old transgenic p GFP-c-Kit mouse, in which all oocytes express GFP-c-Kit green. Nuclei were stained with DAPI grey. Clearly visible are primordial and primary oocytes, as well as the nuclei of surrounding, e.
Specimens of a and b were optically cleared with CUBIC2, the specimen of c was not optically cleared. Microscope: mDSLM. Objective lenses: Epiplan-Neofluar 2. Excitation wavelength: nm. Liver-derived organoids were cultured in expansion medium for seven days as previously described Pancreas-derived organoids were cultured as previously described 18 with adjustments for human pancreas-derived organoids Meritxell Huch, personal communication, unpublished data.
Isolation of organoids from the embedding matrix human liver organoids: Matrigel, Corning; human pancreas organoids: Cultrex BME2, Amsbio for fixation and whole-mount staining was performed with slight modifications of protocols published by Broutier et al. Briefly, organoids were extracted from the matrix by washing three times with ice-cold 0. Whole-mount staining of the specimens was done using an adapted version of a protocol previously published by Smyrek et al.
Liver organoid clusters Fig. These cuvettes with a square cross section were attached to stainless steel pins and pre-filled with PBS. The same mounting procedure was used for single human pancreas organoids Fig. For nuclei segmentation, murine pancreas organoids were grown within ultra-thin FEP-foil cuvettes for three days Supplementary Fig.
For imaging, the filled FEP-cuvettes were attached to stainless steel pins using instant glue. Figure 3 Projections and fusion of image stacks of human liver organoids recorded along four directions for multiple-view fusion. A cluster of human liver organoids in an ultra-thin square cross section FEP-foil cuvette. Four stacks consisting of images each were recorded with a DSLM along four different directions, i.
The projections for each rotation angle are aligned to show the specimen along a comparable orientation. Arrows indicate the orientations of the light paths of excitation blue and detection green for each angle. Cell nuclei DAPI. Full size image Live specimens Live murine organoids in FEP-foil cuvettes: Murine pancreas organoids were also grown within ultra-thin FEP-foil cuvettes for up to 10 days. Figure 4 Long-term culture of murine pancreas organoids fixed and labelled inside ultra-thin FEP-foil cuvettes.
Vujinn Transmission of multiple system atrophy prions to transgenic mice. Peripheral administration of tau aggregates triggers intracerebral tauopathy in transgenic mice. In the case no disassembly occurs within the cleaning solution, the latter needs to be handled as a biohazard while no such requirement is needed in the case of complete disassembly. None of the fibrillar assemblies listed and used in this work has been demonstrated to be infectious in humans. Efficiency of different cleaning hellmwnex to remove amyloid fibrillar assemblies from plastic, glass, aluminum and stainless steel surfaces. Composition — Hellma Analytics Colors spanning from brown high to dark green low integrate the proportion of material remaining adsorbed and of fibrillar nature. Download Hellmanex Safety Data Sheet Exogenous alpha-synuclein fibrils induce Lewy Body pathology leading to synaptic dysfunction and neuron death.
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