The changes in intensity with time for each tile was fitted to a one-phase association function (\({\rm{f}}({\rm{x}})={y}_{0}+(p-{y}_{0})\cdot [1-exp(-x/\tau )]\))6 to get the maximum values of transparency (P; plateau) and time constant () of diffusion. Fast extraction occurred for 13hr of ETC (thick red line in Fig. were transcardially perfused with PBS and paraformaldehyde (PFA), and fixed organ samples were dissected and incubated in hydrogel solution overnight at 4C. Because ECM arrangements are unique signatures of each organ, these results indicate that organs have unique features of tissue clearing based on their protein composition, ECM contents, and ECM arrangements. Tissue-clearing techniques have received great attention for volume imaging and for the potential to be applied in optical diagnosis. See all available courses Continuing Education Credits P.A.C.E. Contact Hours (acceptable for AMT, ASCP, and state recertification): 1 hour (s) Approved through 8/31/2024 Susaki, E. A. et al. In summary, multiple factors affecting tissue transparency during the tissue-clearing process of the brain, liver, kidney, and lung were comprehensively investigated, and how the specific characteristics of different organ tissues contribute to the reduction of light scattering or the improvement of tissue porosity was clarified. b. 1), and we chose 1-mm thickness for further analyses. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. a supply of clean, filtered paraffin wax held at 2-4C above its melting point. Briefly, 7-week-old C57BL/6 mice (DAEHAN Biolink, Inc, Korea.) Fig. - temp - vacuum and pressure - agitation. To equalize concentrations inside and outside blocks of tissue this depends on Ficks Law: the rate of solution diffusion through tissues is proportional to the concentration gradient (the difference between the concentrations of the fluids inside and outside the tissue) as a multiple of temperature dependant constants for specific substances. The changes in local transparency of each tile are then automatically calculated to deduce the maximum transparency and time constant () by fitting the time series to the one-phase association function (Fig. CAS Therefore, this is one of the major leading issues for biobanking guidelines and best practices [1,2,3,4,5] and it will also form the backbone for evaluating how and where to freeze tissue samples, as discussed in this chapter. 5). Article Resin is used exclusively as the embedding medium for electron microscopy, ultra-thin sectioning for high resolution and also for undecalcified bone. Cite this article. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. Factors that Impact Tissue Embedding. Li J, Smyth P, Cahill S, Denning K, Flavin R, Aherne S, Pirotta M, Guenther SM, O'Leary JJ, Sheils O. BMC Biotechnol. Opt Lett 39, 28882891, https://doi.org/10.1364/OL.39.002888 (2014). J Microsc-Oxford 242, 148156, https://doi.org/10.1111/j.1365-2818.2010.03448.x (2011). Impregnation time for dense fatty tissue can be greatly reduced with the addition of vacuum during processing. Fig. Penetration. 4a. Nat Neurosci 14, 14811488, https://doi.org/10.1038/nn.2928 (2011). Cleared specimens are often subject to further processing for optical examinations with fluorescent labeling of specific macromolecules1,3,4,12,13,14. https://doi.org/10.1016/j.cell.2014.10.010, https://doi.org/10.1016/j.cell.2014.03.042, https://doi.org/10.1111/j.1365-2818.2010.03448.x, https://doi.org/10.1016/j.cell.2015.06.067, https://doi.org/10.1016/j.expneurol.2015.03.020, https://doi.org/10.1016/0006-8993(92)91000-5, https://doi.org/10.1038/s41598-018-26776-9, https://doi.org/10.1016/j.ces.2004.04.028, https://doi.org/10.1117/1.Jbo.22.9.091506, https://doi.org/10.1016/j.cell.2014.10.034, https://doi.org/10.1016/j.celrep.2016.06.060, https://doi.org/10.1016/j.celrep.2017.06.010, https://doi.org/10.1038/s41551-017-0139-0, https://doi.org/10.1016/j.neuroscience.2012.06.059, https://doi.org/10.1371/journal.pone.0154942, http://creativecommons.org/licenses/by/4.0/, Dynamic interaction of injected liquid jet with skin layer interfaces revealed by microsecond imaging of optically cleared ex vivo skin tissue model, Optimized single-step optical clearing solution for 3D volume imaging of biological structures, Optical clearing and testing of lung tissue using inhalation aerosols: prospects for monitoring the action of viral infections, An Optimized Mouse Brain Atlas for Automated Mapping and Quantification of Neuronal Activity Using iDISCO+ and Light Sheet Fluorescence Microscopy. You are using a browser version with limited support for CSS. Factors Affecting Tissue Processing X marked points in (d) indicate an optimal time point for each organ for maximal clearing. 3). Chen, B. C. et al. Because the composition and structure of tissues are different, the contributions of these factors on tissue clearing are also tissue-specific. We also found that the kinetics of lipid extraction is not linear, and at least two different extraction kinetics of DiI-labeled lipid components were recognized, although the exact identification of these molecules needs to be further explored. Robel, S., Berninger, B. After devitalization of the specimen, there is a variable length of time before the specimen is placed in fixative. Fixation - factors affecting fixation. MeanSD for tiles of normal area at TBI days 3, 7, 14 (N=37, 71, 68 respectively) and injury area at TBI days 3, 7, 14 (N=89, 54, 46 respectively). 3c (kidneyTissue Processing document.getElementById( "ak_js_1" ).setAttribute( "value", ( new Date() ).getTime() ); You have entered an incorrect email address! 3a). However, industrial hygiene experiences from micron-scale particles may . A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Pan, C. C. et al. Specimen geometry. Furthermore, the extraction profile of other lipid species labeled with Oil-Red-O (ORO) was markedly different from that of DiI-labeled lipids (Suppl. The collagen contents of samples were quantified using a Sircol collagen assay kit (Biocolor, Carrickfergus, UK) according to the manufacturers instructions. We analyzed RNA quality using, RNA quality and gene expression profiles from different fixative buffers. Fig. This step was done following collagen extraction by incubation for overnight in a pepsin-containing solution at 65C. Automated processors incorporate vertical or rotary oscillation. The smaller the size of the molecules in the solution, the faster the rate of fluid penetration (low viscosity) and vice versa. Nat Protoc 9, 16821697, https://doi.org/10.1038/nprot.2014.123 (2014). The first stage of processing is the removal of free unbound water and aqueous fixatives from the tissue components. Coagulation is caused by the dehydration of proteins through the use of alcohols or acetone. 1f, illustrating the differences in the tissue-clearing properties. Thus, the homogeneity of the component should increase, and the light scattering should be reduced. For experiments on warm ischemia, RNA was assayed before fixation and processing. (SPIE Press, 2006). Total RNA was extracted from mouse kidney. Write CSS OR LESS and hit save. Hard tissue can be immersed in a glycerol/alcohol mixture. On the other hand, we propose that optical properties after tissue clearing per se can be used for the label-free assessment of tissue conditions and an optical diagnosis. Unable to load your collection due to an error, Unable to load your delegates due to an error. To investigate the effect of density and structure of ECM on tissue transparency, finite difference time domain (FDTD) simulation was performed. Understanding Preanalytical Variables and their Effects on Clinical A guide to light-sheet fluorescence microscopy for multiscale imaging. d. Temperature. Indeed, a shift of the spots from the lower-right to upper-left side was observed in tissues with the progression of ETC (Fig. If the concentration gradient is excessive diffusion currents across the cell membranes may increase the possibility of cell distortion. Cell 157, 726739, https://doi.org/10.1016/j.cell.2014.03.042 (2014). Diagnostic application of tissue clearing (a) Images of mouse brain 3, 7, and 14 days after cryogenic TBI. Heat increases the rate of penetration and fluid exchange. Fluids with a low boiling point are generally more readily replaced, Viscosity influences the speed of penetration of the clearing agent, Amyl acetate, methyl benzoate and methyl salicylate, Flammable and colorless liquid with a characteristic petroleum odour. These results indicate that ECM deposition occurred in the injured area, which can be captured by the currently developed procedure (Fig. Representative data are presented as an electropherogram (, RNA quality and gene expression profiles from different fixative buffers. Three-Dimensional Study of Alzheimers Disease Hallmarks Using the iDISCO Clearing Method. Sci Rep 6, 32674, https://doi.org/10.1038/srep32674 (2016). HHS Vulnerability Disclosure, Help They are extremely oily and cannot be recycled. We greatly appreciate the thoughtful comments from Dr. Jae Ryun Ryu and Hyun Jung Kim, technical support from the members of Suns lab, and drawing of the schematic figures by Ye-Jin Son. Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue. 2). CAS Similarly, enhanced optical contrast with fibrosis was also found in the focal lung fibrosis model (Suppl. 2022 Jun 10;10:792847. doi: 10.3389/fpubh.2022.792847. Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping. Fig. For this reason, measuring tissue porosity is important, but an appropriate method is currently lacking. Many dehydrating reagents are hydrophilic and interact with the water molecules in the tissue by hydrogen bonding. Factors such as specimen size and thickness are determined during the collection and tissue preparation or grossing phase, which the laboratory typically has very little influence over. Following fixation in alcoholic fixatives such as Carnoys fluid dehydration can be initiated in 100% ethanol. The mold is placed on a small cooling area to allow the paraffin. However, because extended ETC will progressively remove DiI signal from weakly labeled capillaries, caution is necessary for using this technique for high-fidelity, quantifiable analysis. Tissue Processing made easy and interesting Thanks. Unauthorized use of these marks is strictly prohibited. Google Scholar. ISSN 2045-2322 (online). This manuscript contains online supplemental material at http://www.jhc.org. By submitting a comment you agree to abide by our Terms and Community Guidelines. how does temperature affect tissue processing? In fact, the homogeneous diffusion of antibody deep into a thick tissue or whole organ is crucial for imaging the specific target labeled by a fluorescence protein. The darker gray color represents conditions associated with better RNA quality from FFPE tissue. The contribution of RI-matching to tissue transparency increased in large-size-change tissue (brain, liver) by ETC, but decreased in small-size-change tissue (kidney, lung) (Suppl. . Vacuum will remove reagents from the tissue but only if they are more volatile than the reagent being replaced. Article Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials. Accessibility Gelatin is primarily used in the production of sections of whole organs using the Gough-Wentworth technique and in frozen sectioning. The Impact of Pre-analytical Quality Initiatives on Cholangiocarcinoma Diagnostics in Thailand. The tissue was immersed in 100% dimethyl sulfoxide (DMSO) for enough time to improve the homogenous loading DiI deep into the tissue, after which it was again immersed in 0.2mM DiI overnight. (e) Contributions of RI-matching and lipid extraction to tissue clearing, if the effect of size change on transparency is compensated. and JavaScript. Rate of reaction. ADS 5. 3 factors affecting tissue processing? (f) 3D plot of contributions of lipid-extraction and RI-matching to tissue transparency. Alcohol Most common dehydrating agent - Removes bound water creating dipeptide -Over dehydration cannot be reversed (results in chattered sections) ethanol -Strictly controlled by federal government -Clear, colorless, flammable liquid -Reliable and fast acting - 60-70%, 80 %, 95%, 100% concentrations used to gradually remove H20 Efficient agitation may reduce the processing time up to 30%. Therefore, the complete removal of lipid species is not achievable when using an SDS-based protocol, and organic solvent-based methods such as DISCO series2,23,24 or CUBIC3 should be considered. Sci Rep 8, 12815 (2018). Most commonly used clearing agent in routine histology and is also recyclable. Accordingly, de-lipidation causes tissue hydration and swelling, while optical clearing reagent reverses it via water efflux. Provided by the Springer Nature SharedIt content-sharing initiative. Chung JY, Kim K, Ylaya K, Walker-Bawa KE, Perry C, Star RA, Hewitt SM. ADS Thus, this trade-off in lipid extraction effects seems to differentially influence the tissue-clearing process. For measurements, transparency was acquired by fitting with the one-phase association function [see Methods section Time constant () of diffusion measurement]. N=3, 3, 6, 6 for brain, lung, liver and kidney, respectively. We re-defined the ACT process into 5 steps as shown in Fig. And for infinite area and finite thickness of virtual ECM structure, a periodic boundary condition was applied along the x-axis and, a perfectly matched layer was applied along y-axis. Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Relate how quality control measures ensure consistent tissue processing and identify tissue that is poorly processed and state a remedy for the problem. (b) Correlation between the actual collagen amount and variation of transparency on different levels of RI-matching. Internet Explorer). DiI [(2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole; perchlorate] is commonly used for visualizing lipids in the cell membrane. The slices were imaged using a conventional camera before and after clearing. However, a size change is not desirable in many applications because of the distortion caused by anisotropic expansion/shrinkage, and there are modified versions of tissue clearing with adjustments made to the clearing solution to prevent size changes. Tissue Processing in Histology Other reagents affect dehydration by repeated dilution of the aqueous tissue fluids. CAS Using pressure to increase the rate of infiltration decreases the processing time. For this reason specimens are processed through a graded series of reagents of increasing concentration. For the experiments in this study, the variables were studied in isolation. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. 4b). 2022 Aug;12(8):160. doi: 10.1007/s13205-022-03223-1. Most clearing agents are hydrocarbons with high refractive indices (approaching that of dehydrated fixed tissue protein) and, on immersion, anhydrous tissues are rendered transparent or clear similar to protein so they are termed as clearing agent. Finally, we tested whether changes in the optical status of tissues during the clearing procedure could be used to evaluate tissue quality under pathological conditions. Duvuru Prathiba Sri Ramachandra University Abstract Introduction: Quality monitoring in histopathology unit is categorized into three phases, pre-analytical, analytical and post-analytical, to. Hama, H. et al. Article Such variables include the interval between cellular death and fixation. Based on this notion, recently we demonstrated that complete drying of ACT-processed samples can transform them into film-like, highly transparent thin sheet, which can be used to reduce imaging burden26. Decalcification technique helps to remove calcium salt from the tissue without affecting the morphology of the tissue and staining. To induce cryogenic TBI, a metal probe with a diameter of 5mm was cooled in liquid nitrogen. e. Concentration. (d) Finite-difference time-domain (FDTD) simulation with randomly generated images of various ECM density showing that light transmittance is dependent on ECM density (from negative slop of the linear fitting line) and ECM structures (from red points having similar ECM density). ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. (v) To facilitate the establishment of tissue banks in other parts of the country as an integrated branch of the hospital. 1 Citations Abstract This chapter discusses the method of decalcification of bony and hard tissue for histopathology processing. Fig. Because ECM architecture cannot be experimentally modified, a finite-difference time-domain (FDTD) simulation with randomly generated images (Suppl. Enter your email address and name below to be the first to know. 4g). Kim, J.H., Jang, M.J., Choi, J. et al. Tissue sample quality is crucial not only when tissues are used for diagnostics development but also for medical research. These reagents remove and replace free water in cells and tissues and cause a change in the tertiary structure of proteins by destabilizing hydrophobic bonding. The head skin of anesthetized mice was cut, and the pre-chilled metal probe was placed in contact with the cranium of the mice for 30seconds, as described previously38. To evaluate the effect of size change on transparency of cleared tissues independently without RI-matching, it is crucial that the refractive index of the solutions used for changing size of tissue have to be preserved. Article PubMed Central DEHYDRATION The first stage in tissue processing is dehydration (the removal of water). Excessive dehydration may cause the tissue to become hard, brittle and shrunken. The profiling of total RNA extracted from warm ischemia time conditions by microcapillary electrophoresis. We additionally compared the tissue size and transparency in various concentrations of PBS (0.110) and found that the correlation between size change and transparency was very similar in all organs (Suppl. The relationships of the contributions of lipid extraction and RI matching to the final transparency of different organs are shown in Fig. Lee, E. et al. 1c,d). Choice of a dehydrant is determined by the nature of the task, the embedding medium, processing method, and economic factors. Hewitt SM, Lewis FA, Cao Y, Conrad RC, Cronin M, Danenberg KD, Goralski TJ, Langmore JP, Raja RG, Williams PM, Palma JF, Warrington JA. Jao, C. Y., Roth, M., Welti, R. & Salic, A. Metabolic labeling and direct imaging of choline phospholipids in vivo. Several factors have influence on staining intensity (Box 7.2): 1. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. Ex vivooptical measurements of glucose diffusion kinetics in native and diabetic mouse skin. PubMed Central Type of processing and the processor system to be used, Processing conditions such as temperature, vacuum and pressure, The boiling point of the clearing agent gives an indication of its speed of replacement by melted paraffin wax. Furthermore, our protocols can be used to verify the pathological changes of tissues, which will be a foundation for the label-free diagnosis of tissues based on optical clearing methods. BrainFilm, a novel technique for physical compression of 3D brain slices for efficient image acquisition and post-processing. Bookshelf Tissue Fixation and Fixatives | Research at St. Michael's Hospital First, the glass bottom of a 35-mm-diameter black confocal dish (102350, SPL, Republic ofKorea) was covered with a thin polydimethylsiloxane (PDMS) sheet, onto which a tissue stored in PBS was pinned using a short thin metal wire to prevent it from floating or moving when the solution was exchanged. government site. The specimen undergoes some form of preparation before fixation, depending on the size and complexity of the specimen. Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response. Save my name, email, and website in this browser for the next time I comment. Google Scholar. Polymerized samples were cut into 1-mm-thick tissue slices and electrophoresed for fast removal of lipid, using an ETC apparatus (X-CLARITY, Logos systems, Republic of Korea) with following conditions: 1.5mA, 37C. 3b (brainTissue processing - how to succeed with your IHC For the purpose of tissue processing in the histopatholo-gy, fixation of tissue is considered as necessary and essential step. 2,11-14 Hence, a prospective investigator must consider factors involved in sample processing and tissue acquisition before embarking on a project. Intro to Tissue Fixation in Histology: Types, Methods & More There are a number of factors that will affect the fixation process: Buffering Penetration . and W.S. (b) Time-transparency data fitted to one-phase association function to get time constant and final transparency. DiI (468495, Sigma-Aldrich, USA) is known to bind phospholipid; hence, we used it to measure the amount of phospholipid in a tissue. (d) Size changes of each organ at each tissue-clearing step. Chung, K. et al. PubMed The acidic dye such . Fig. Heating the paraffin wax to a high temperature alters the properties of the wax. (d) Sample images of the same mouse kidney slice: bright-field image (left), transparency map (middle), map (right). 2022 Sep 6;23(18):10267. doi: 10.3390/ijms231810267. These include: sample thickness, tissue orientation, quality of paraffin wax, mold and cassette types, and . Tissue Preservation and Factors Affecting Tissue Quality 2.1. 5). Thus, satisfactory optical clearing can be achieved by simple RI adjustment in some tissues for which the lipid contribution is low. PubMedGoogle Scholar. 2b. Longer tissue processing times were associated with higher quality RNA. An interactive meta-analysis of MRI biomarkers of myelin - PMC 4c). 57, https://doi.org/10.1364/ao.57.004839 (2018). After tissue processing, the specimen is embedded (surrounded with paraffin that acts as a solid support for microtomy). official website and that any information you provide is encrypted It is inexpensive and provides quality sections, Compatible with most routine and special stains, These additives helps to increase hardness. Efficient agitation may reduce the processing time up to 30%. The recent explosion of tissue-clearing techniques, which have been successfully applied to glimpse whole or parts of body structures, reflects this demand1,2,3,4. PLoS One 11, e0154942, https://doi.org/10.1371/journal.pone.0154942 (2016). & Tuchin, V. V. Recent progress in tissue optical clearing. Methods Mol Biol. Examples include Dioxane, tertiary butanol and tetrahydrofuran. Cleared tissues were immersed in ORO (2mM in 100% isopropanol, 10L/mg) and incubated at room temperature for 2hours. (June 2008) Types of fixation and processes [ edit] There are generally three types of fixation processes depending on the sample that needs to be fixed. For that, we used different concentration of PBS (0.1, 0.5, 1, 5 and 10) and 4% SDS, whose refractive indices were 1.3328, 1.3331, 1.3343, 1.3397, 1.3468 and 1.3339 respectively. The contributions of factors to tissue transparency were calculated the difference in transparency between the two different steps. For normal routine work 76 25 mm slides are universally used. Thus, it is difficult to remove these slow components of lipids completely using SDS-based ETC methods, and extended ETC (>24hr) might cause tissue damage.