FDISCO+: a clearing method for robust fluorescence preservation of cleared samples
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ABSTRACT:
SUBMITTER: Wan P
PROVIDER: S-EPMC8427119 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The development of tissue clearing technologies allows 3D imaging of whole tissues and organs, especially in studies of the central nervous system innervated throughout the body. Although the three-dimensional imaging of solvent-cleared organs (3DISCO) method provides a powerful clearing capacity and high transparency, the rapid quenching of endogenous fluorescence and peroxide removal process decreases its practicability. This study provides a modified method named tDISCO to solve these limitations. The tDISCO protocol can preserve AAV-transduced endogenous EGFP fluorescence for months and achieve high transparency in a fast and simple clearing process. In addition to the brain, tDISCO was applied to other organs and even hard bone tissue. tDISCO also enabled us to visualize the long projection neurons and axons with high resolution. This method provides a fast and simple clearing protocol for 3D visualization of the AAV- transduced long projection neurons throughout the brain and spinal cord.
| S-EPMC9455935 | biostudies-literature
Project description:Tissue optical clearing techniques provide alternative approaches for imaging large-volume specimens. uDISCO, an organic-solvent-based method, stands out from the enormous array of available optical clearing methods by achieving whole-brain imaging with high transparency, size reduction and fluorescence preservation. In this study, we aimed to modify the uDISCO protocol to achieve better fluorescence preservation and to thereby further improve its optical imaging quality. First, we determined the optimal pH value for optimized uDISCO, termed "a-uDISCO" (alkaline pH-based uDISCO). Then, we compared fluorescence preservation between a-uDISCO and uDISCO. In addition, we validated the clearing performance of the optimized method according to several parameters, including tissue transparency, size changes, and the maintenance of cell morphology. Finally, we demonstrated that a-uDISCO enabled the high-quality brain-wide visualization of neuronal structures. This method potentially provides a better alternative for high-throughput imaging of samples with low-level fluorescence protein expression or for archiving and repetitive revisiting of rare samples.
| S-EPMC6104128 | biostudies-literature
Project description:UnlabelledPremise of the studySeveral recent waves of interest in leaf architecture have shown an expanding range of approaches and applications across a number of disciplines. Despite this increased interest, examination of existing archives of cleared and mounted leaves shows that current methods for mounting, in particular, yield unsatisfactory results and deterioration of samples over relatively short periods. Although techniques for clearing and staining leaves are numerous, published techniques for mounting leaves are scarce. •Methods and resultsHere we present a complete protocol and recommendations for clearing, staining, and imaging leaves, and, most importantly, a method to permanently mount cleared leaves. •ConclusionsThe mounting protocol is faster than other methods, inexpensive, and straightforward; moreover, it yields clear and permanent samples that can easily be imaged, scanned, and stored. Specimens mounted with this method preserve well, with leaves that were mounted more than 35 years ago showing no signs of bubbling or discoloration.
| S-EPMC4162665 | biostudies-literature
Project description:We provide here a procedure enabling light sheet fluorescence microscopy (LSFM) of entire human eyes after iDISCO + -based clearing (ClearEye) and immunolabeling. Demonstrated here in four eyes, post-processing of LSFM stacks enables three-dimensional (3D) navigation and customized display, including en face viewing of the fundus similarly to clinical imaging, with resolution of retinal capillaries. This method overcomes several limitations of traditional histology of the eyes. Tracing of spatially complex structures such as anterior ciliary vessels and Schlemm's canal was achieved. We conclude that LSFM of immunolabeled human eyes after iDISCO + -based clearing is a powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.
| S-EPMC10564773 | biostudies-literature
Project description:BackgroundTissue-clearing techniques have recently been developed to make tissues transparent for three-dimensional (3D) imaging at different scales, including single-cell resolution. However, current tissue-clearing workflows have several disadvantages, including complex protocols, time-consuming application, and fluorescence quenching. Additionally, they can be used mainly for clearing larger-volume samples, preventing wide and easy applicability in conventional experimental approaches. In this study, we aimed to develop a versatile, fast, and convenient method for clearing thin and semi-thick samples, which can be used for three-dimensional imaging of experimental or even clinical samples.ResultsWe developed an alkaline solution (AKS) containing a combination of 2,2'-thiodiethanol (TDE), DMSO, D-sorbitol, and Tris for tissue clearing, as the alkaline environment is suitable for maintaining the fluorescence of most commonly used fluorescence protein GFP and its variants, and tested its clearing effect on samples from mice and human brains. We assessed the clearing speed, the preservation of fluorescence protein and dyes, and the imaging depth and quality. The results showed that AKS treatment rapidly cleared 300-μm-thick brain slices and 1-mm-thick slices from different organs within 5 min and 1 h, respectively. Moreover, AKS was compatible with a variety of fluorescence proteins and dyes. Most importantly, AKS enhanced the fluorescence of YFP, in contrast to the majority of existing tissue-clearing methods which reduce the fluorescence intensity of fluorescent proteins. Using AKS, we performed long-time high-resolution imaging of weak fluorescent protein-labelled tissues, long-distance fibre tracking, larger-scale 3D imaging and cell counting of the entire brain area, neural circuit tracing, 3D neuromorphic reconstruction, and 3D histopathology imaging.ConclusionsAKS can be used for simple and rapid clearing of samples from mice and human brains and is widely compatible with a variety of fluorescent dyes. Therefore, AKS has great potential to be used as a broad tissue-clearing reagent for biological optical imaging, especially for time-sensitive experiments.
| S-EPMC8966190 | biostudies-literature
Project description:Over the last two decades, several tissue clearing methodologies have been established that render tissues optically transparent and allow imaging of unsectioned tissues of significant volumes, thus improving the capacity to study the relationships between cell and 3D tissue architecture. Despite these technical advances, the important unsolved challenges that these methods face include complexity, time, consistency of tissue size before and after clearing, and ability to immunolabel various antibodies in cleared tissue. Here, we established very simple and fast tissue clearing protocol, FxClear, which involves acrylamide-free electrophoretic tissue clearing (ETC). By removal of the acrylamide infusion step, we were able to achieve fast reaction time, smaller tissue expansion, and higher immunoreactivity. Especially, immunoreactivity and fluorescence intensity were increased in FxClear-processed tissues compared to un-cleared tissues. Our protocol may be suitable for small-sized biopsy samples for 3D pathological examinations.
| S-EPMC6614074 | biostudies-literature
Project description:Resin embedding is widely used to dissect the fine structure of bio-tissue with electron and optical microscopy. However, it is difficult to embed large-volume tissues with resin. Here, we modified the formula of LR-White resin to prevent the sample cracking during polymerization process and applied this method to the intact brains of mouse, ferret, and macaque. Meanwhile, we increased the fluorescence preservation rate for green fluorescent protein (GFP) from 73 ± 4.0% to 126 ± 3.0% and tdTomato from 60 ± 3.3% to 117 ± 2.8%. Combined with the whole-brain imaging system, we acquired the cytoarchitectonic information and the circuit information such as individual axon and boutons which were labeled with multiple fluorescent proteins. This method shows great potential in the study of continuous fine microstructure information in large-volume tissues from different species, which can facilitate the neuroscience research and help the understanding of the structure-function relationship in complex bio-tissues.
| S-EPMC7645060 | biostudies-literature
Project description:Investigation of the local sample elasticity is of high importance in many scientific domains. In 2014, Herruzo et al. published a new method based on frequency-modulation atomic force microscopy to locally determine the elasticity of samples (Nat. Commun.2014, 5, 3126). This method gives evidence for the linearity of the relation between the frequency shift of the cantilever first flexural mode ?f1 and the square of the frequency shift of the second flexural mode ?f22. In the present work, we showed that a similar linear relation exists when measuring in contact mode with a certain load FN and propose a new method for determining the elastic modulus of samples from this relation. The measurements were performed in non-dry air at ambient temperature on three different polymers (polystyrene, polypropylene and linear low-density polyethylene) and a self-assembled monolayer of 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) on a silicon oxide substrate perforated with circular holes prepared by polymer blend lithography. For all samples the relation was evidenced by recording ?f1, ?f2 and FN as a function of the Z-displacement curves of the piezoelectric scanner. The occurence of a plastic deformation followed by an elastic deformation is shown and explained. The necessary load FN for measuring in the elastic domain was assessed for each sample, used for mapping the frequency shifts ?f1 and ?f2 and for determining the elastic modulus from ?f22/?f1. The method was used to give an estimate of the Young's modulus of the FDTS thin film.
| S-EPMC5769082 | biostudies-other
Project description:Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.
| S-EPMC7253160 | biostudies-literature
Project description:BackgroundIn cell biology, increasing focus has been directed to fast events at subcellular space with the advent of fluorescent probes. As an example, voltage sensitive dyes (VSD) have been used to measure membrane potentials. Yet, even the most recently developed genetically encoded voltage sensors have demanded exhausting signal averaging through repeated experiments to quantify action potentials (AP). This analysis may be further hampered in subcellular signals defined by small regions of interest (ROI), where signal-to-noise ratio (SNR) may fall substantially. Signal processing techniques like blind source separation (BSS) are designed to separate a multichannel mixture of signals into uncorrelated or independent sources, whose potential to separate ROI signal from noise has been poorly explored. Our aims are to develop a method capable of retrieving subcellular events with minimal a priori information from noisy cell fluorescence images and to provide it as a computational tool to be readily employed by the scientific community.ResultsIn this paper, we have developed METROID (Morphological Extraction of Transmembrane potential from Regions Of Interest Device), a new computational tool to filter fluorescence signals from multiple ROIs, whose code and graphical interface are freely available. In this tool, we developed a new ROI definition procedure to automatically generate similar-area ROIs that follow cell shape. In addition, simulations and real data analysis were performed to recover AP and electroporation signals contaminated by noise by means of four types of BSS: Principal Component Analysis (PCA), Independent Component Analysis (ICA), and two versions with discrete wavelet transform (DWT). All these strategies allowed for signal extraction at low SNR (-?10?dB) without apparent signal distortion.ConclusionsWe demonstrate the great capability of our method to filter subcellular signals from noisy fluorescence images in a single trial, avoiding repeated experiments. We provide this novel biomedical application with a graphical user interface at https://doi.org/10.6084/m9.figshare.11344046.v1 , and its code and datasets are available in GitHub at https://github.com/zoccoler/metroid .
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