The LS&E Microscopy core facility the invites You to attend special seminars in Advanced Microscopy Application and Methodologies.
The next seminars will be on “Clearing methodologies for biological samples“.
Date: Tuesday, April 25th, 10:30 o’clock, (45 min’s talk)
Location: Room 4-17, Emersson Building
Speaker: Shlomi Lazar, Ph.D.
Division of medical chemistry, The Israel institute for Biological Research (IIBR).
Title: Looking inside the organs: a comparative analysis of three methods for establishing optically transparent samples
Typical histological study, using sectioning of the tissue, has major limitations in obtaining 3D images of structural components and cells distribution within tissues. Ideally, samples should be imaged at high spatial resolution with minimal sectioning. However, thick tissue imaging is limited mostly because of light scattering. In this study, we compared the efficacy of three recently published clearing protocols (Scale, 3DISCO and Clarity) in generating a transparent thick section which can be subjected to confocal analysis.
Brains, ovaries and embryos obtained from transgenic mice expressing enhanced yellow fluorescent protein (eYFP) in specific cells or wild type mice, were collected and cut to thick sections (2-3mm). Some slices were first fluorescently immunolabeled for various markers. All sections were processed in one or more of the following protocols: (a) The Scale protocol was based on aqueous reagent that renders biological samples optically transparent and completely preserves fluorescent signals in the clarified structures; (b) 3DISCO (3D imaging of solvent cleared organ) was based on organic solvents that dissolve the lipid structure in the tissue and (c) The Clarity protocol was based on hydrogel tissue embedding, lipid extraction in aqueous solution, and clearing. All sections were subjected to confocal analysis. Data acquired was processed using ImageJ software.
We successfully optimized immunofluorescence labeling protocol for floating thick sections (2-3mm). Evaluation of the clear tissues produced by each of the three methods showed that Scale protocol resulted in fragile and only slightly cleared sections. 3DISCO method demonstrated transparent slices but a degraded fluorescent signal over time (1 day half-life). Thick slices processed by Clarity protocol were nicely transparent, maintained their fluorescence intensity, but were greatly more expensive. A combination of Clarity and Scale protocols replacing the high cost clarity reagent with Scale solution, improved the outcome of Scale protocol and preserved the required fluorescence intensity. All slices were successfully analyzed using confocal microscope and no further sectioning was needed.
A combined protocol of Clarity and Scale provide a low cost and available way to generate transparent organs. This enabled detailed analysis of structural and molecular information from thick organ samples.
Date: Tuesday, April 25th, 11:30 o’clock, (20 min’s talk)
Location: Room 4-17, Emersson Building
Speaker: Nadav Yayon, M. Sc
PhD student at Prof. Hermona Soreq’s lab, The Edmond and Lily Safra Center of Brain Science. The Hebrew University of Jerusalem
Title: Extracting micro-scale features from macro-scale imaging of iDISCO brains
Recent technological advancements enable rendering samples transparent and imaging huge areas of tissue rapidly and in high resolution. However, while these technologies produce beautiful images and 3D movies, extracting quantitative information from such experimental samples is confronted with significant difficulties, both due to sample preparation procedures and because of imaging and analysis constraints. In my talk, I will share my personal experience of using the iDISCO clearing approach and Light-sheet imaging, and will describe our attempts to accurately extract 3D information to identify the location, density and morphology of cortical interneurons in the mouse cortex.
Light refreshment will be served before the seminar
Registration below is free of charge but required due to limited place.
Looking forward to seeing you all,
LSE Microscopy team