Masselink W et al. (2019), Broad applicability of a streamlined ethyl cinn...

XB-ART-55645

Development 2019 Feb 01;1463:. doi: 10.1242/dev.166884.

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Broad applicability of a streamlined ethyl cinnamate-based clearing procedure.

Masselink W , Reumann D , Murawala P , Pasierbek P , Taniguchi Y , Bonnay F , Meixner K , Knoblich JA , Tanaka EM .

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Turbidity and opaqueness are inherent properties of tissues that limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here, we describe 2Eci (2nd generation ethyl cinnamate-based clearing), which can be used to clear a wide range of tissues in several species, including human organoids, Drosophila melanogaster, zebrafish, axolotl and Xenopus laevis, in as little as 1-5 days, while preserving a broad range of fluorescent proteins, including GFP, mCherry, Brainbow and Alexa-conjugated fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method opens up tissue clearing to a much broader group of researchers due to its ease of use, the non-toxic nature of ethyl cinnamate and broad applicability.

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Species referenced: Xenopus laevis
Genes referenced: col1a2 grap2 mylkl prrx1 sox2

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	Fig 1 Ethyl cinnamate clearing optimizations in cerebral organoids. Whole-mount recording of >100-day-old cerebral organoids after fixation without clearing (A), using FluoClearBABB (B) and ethanolpH9/Eci (C). Yellow arrows mark organoids, two independent repetitions were performed. (D-H) Dehydration agent-dependent fluorescence after Eci-mediated clearing using confocal z-stack recordings. (I) The mean±s.e.m. of the maximal fluorescence of z-stacks was quantified for organoids live mounted in PBS (D) and compared with methanol (E), ethanol (F), 4-butanol (G) and 1-propanol (H) dehydration (30%, 50%, 70%, 2×100% at pH 9.0) and subsequent refractive index match with Eci. (n=6). Data are mean±s.d. P<0.05, **P<0.001. (J) Quantification of tissue autofluorescence (GFP− organoids) using confocal z-stack recordings through alcohol-Eci cleared organoids as a measure of clearing efficiency relative to maximum intensity. Data are mean±s.d. Uncleared organoids (K) are efficiently cleared (L) in as little as 25 h, including fixation, dehydration/delipidation and refractive index matching. (M) Schematic illustration showing the timeline of fixation, dehydration and RI matching required for 2Eci implementation on cerebral organoids. Scale bars: 3 mm in A-C,K,L; 100 µm in D-H. Significance was calculated using one-way ANOVA and a post-hoc Tukey's test.
	Fig 2 Characterization of 2Eci clearing in human cerebral organoids. (A,B) Colour-coded z-projection and representative z-slices of both 2Eci cleared (A) and uncleared (B) sparsely labelled (3% GFP+) 80-day-old cerebral organoids. 2Eci allows imaging through whole organoids. Spots of colour aggregation depict aggregations of GFP+ neuronal stem cells in neuronal rosettes, whereas maturing neurons distribute more equally throughout the organoid. (C) Detailed morphology can be observed, including neuronal rosettes (red arrowhead) and more mature neurons in a 80-day-old sparsely labelled cerebral organoid. (D-F) 3D reconstruction of multiple neurons in 80-day-old sparsely labelled cerebral organoids. (D,E) Cellular details such as cell body shape and neurites can be observed. (E) Putative dendrites (green arrowheads) and axons (red arrowheads) are maintained after clearing. (E′) Magnified view of the boxed area in E reveals putative dendritic spines (yellow arrowheads). (F) Putative axonal boutons (yellow arrows) can be identified. (G,G′) Colour-coded z projections of both endogenous GFP (G) and GFP antibody staining using primary and secondary antibodies (αGFP568nm) (G′) in a single organoid reveal full penetration of antibodies. (H-K) Antibody labelling for a variety of neuronal markers efficiently labels different populations of cells in cerebral organoids. (H) Colour-coded z projection of a SOX2 antibody-labelled 60-day-old cerebral organoid. SOX2 is a marker for neuronal stem cells, and labels neuronal rosettes in cerebral organoids. (I) Higher magnification of a SOX2+ neuronal rosette in a cerebral organoid. (J,K) Staining for dorsal forebrain progenitors (J) (FOXG1+, PAX6+) as well as early (CTIP2+) and late (SATB2+) born cortical neurons (K). The lumen of the neuronal rosette is marked with a yellow dashed line; the ventricular zone is marked with a white dashed line. Scale bars: 500 µm in A,B; 50 µm in C; 10 µm in D-F; 500 µm in G-H; 50 µm in I-K.
	Fig 3 Axolotl adult and embryos tissues are efficiently cleared using 2Eci. (A-C) Axolotl limb of Prrx1:ER-Cre-ER CAGGs:LP-GFP-LP-Cherry double transgenic animals are efficiently cleared, preserving both GFP (A, green) and mCherry (B, magenta) after 2Eci clearing of double transgenic axolotl. (C,C′) The box in C marks the elbow. Detailed morphology can be observed throughout the limb, including loose connective tissue, skeletal elements and tendons. (D-G) Col1a2:ER-Cre-ER; CAGGs:LP-GFP-LP-Cherry stained with a Prrx1 antibody. Col1a2 cells are indelibly changed from GFP (D) to mCherry (E) signal. Combining this with antibody staining for Prrx1 (F) allows for the creation of a three-channel image, highlighting the complex heterogeneous nature of connective tissue (G). (D-G) Maximum intensity projections of the entire limb. The box marks the elbow. Single slice z positions of the elbow are shown in D′-G′. (H,I) When combined with depigmentation, 2Eci results in efficiently cleared axolotl embryos. (J) Maximum intensity projection of a double transgenic CAGGs:GFP; CAGGs:mCherryNuc axolotl embryo after antibody labelling for GFP and Cherry. (K-P) Single-plane recordings at various z-depths throughout the embryo depicted in J show recordings up to 750 µm deep can be acquired. Scale bars: 500 µm in A-C,D-G,J-P; 200 µm in C′; 250 µm in D′-G′; 1 mm in H,I.
	Fig 4 Drosophila adults and larvae are efficiently cleared using 2Eci. (A,B) Drosophila larvae and adult Drosophila before (A) and after (B) 2Eci clearing. Red dashed outline indicates one of eight cleared Drosophila larvae in the image (B). (C) Maximum intensity projection of a Krp-GFP Drosophila larvae. Krp-GFP+ salivary glands and fat body tissue can be observed throughout the larvae. (D) Whole Krp-GFP Drosophila virgin z-projection. Green represents GFP fluorescence subtracted by 561 autofluorescence; magenta represents 561 nm autofluorescence. (Right) Colour-coded z projections of both GFP-561AF and 568 nm autofluorescence (AF). (E) Immunostaining for GFP using primary and secondary antibodies (Alexa 568) in SBR-GFP adult Drosophila allows for observation of morphological details after 2Eci clearing. Bright dots (yellow arrows) are bristle follicles. Boxes show where the images in F, H and I are taken from. (F) Z projection of the cardia and anterior midgut (white dashed outline), as well as different populations of the midgut epithelium (box G). (G) In the midgut, diploid (intestinal stem cells, enteroblasts and enteroendocrine cells, yellow arrows) and polyploid (enterocytes, blue arrows) cells can be distinguished based on nuclei size. (H) Polyploid fat body cells. (I) Ovaries can be identified, labelling the entire process of egg chamber maturation, from germinal stem cells to meiosis and egg formation (in the direction of the yellow arrow). Scale bars: 500 µm in C-E; 100 µm in F,I; 10 µm in G; 20 µm in H. Grid size: 5×5 mm squares in A,B.

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