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PEA-CLARITY: 3D molecular imaging of whole plant organs.

Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, Furbank RT, Grof CP - Sci Rep (2015)

Bottom Line: Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration.Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves.Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia.

ABSTRACT
Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

No MeSH data available.


Structurally intact 3D projection of a cleared N. tabacum leaf showing retention of nuclear DNA.CLSM 3D projection of a passively cleared (without cell wall enzyme digestion) N. tabacum leaf showing nuclei stained with propidium iodide (red), and cell walls stained with calcofluor white (green). The 3D projection with epidermal layer cut away is shown in (B) and the x, y, z slices are shown in (A, D) and (C) respectively. The 3D video file can be viewed in supplementary 1.
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f2: Structurally intact 3D projection of a cleared N. tabacum leaf showing retention of nuclear DNA.CLSM 3D projection of a passively cleared (without cell wall enzyme digestion) N. tabacum leaf showing nuclei stained with propidium iodide (red), and cell walls stained with calcofluor white (green). The 3D projection with epidermal layer cut away is shown in (B) and the x, y, z slices are shown in (A, D) and (C) respectively. The 3D video file can be viewed in supplementary 1.

Mentions: Tobacco leaf tissue fixed in hydrogel and cleared in SDS but not enzymatically degraded was stained with propidium iodide (PI, red-nucleus) and calcofluor white (CW, green—cell wall) to assess the retention of DNA and cellular structure (Fig. 2). Using CLSM the whole, intact tobacco leaf disc was imaged and the z-stacks were assembled into a raw, unprocessed 3D slice reconstruction (Fig. 2B). Optical penetration through the entire leaf thickness (~150 μm) was achieved. Vascular, epidermal, palisade mesophyll and spongy mesophyll cell structure was maintained with all nuclei fixed within the cytosol of the cell. Even long trichome protrusions remained perpendicular to the epidermal surface layer across the entire leaf disc (see supplementary 1 for basal trichome orientation; full trichomes were not imaged). Furthermore the staining of nuclei by PI demonstrated the retention of nuclear DNA. This technique also allowed deep optical penetration into the vascular bundle with nucleus of the sieve element / companion cell (SE/CC) clearly defined without the need for sectioning or epidermal peeling.


PEA-CLARITY: 3D molecular imaging of whole plant organs.

Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, Furbank RT, Grof CP - Sci Rep (2015)

Structurally intact 3D projection of a cleared N. tabacum leaf showing retention of nuclear DNA.CLSM 3D projection of a passively cleared (without cell wall enzyme digestion) N. tabacum leaf showing nuclei stained with propidium iodide (red), and cell walls stained with calcofluor white (green). The 3D projection with epidermal layer cut away is shown in (B) and the x, y, z slices are shown in (A, D) and (C) respectively. The 3D video file can be viewed in supplementary 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556961&req=5

f2: Structurally intact 3D projection of a cleared N. tabacum leaf showing retention of nuclear DNA.CLSM 3D projection of a passively cleared (without cell wall enzyme digestion) N. tabacum leaf showing nuclei stained with propidium iodide (red), and cell walls stained with calcofluor white (green). The 3D projection with epidermal layer cut away is shown in (B) and the x, y, z slices are shown in (A, D) and (C) respectively. The 3D video file can be viewed in supplementary 1.
Mentions: Tobacco leaf tissue fixed in hydrogel and cleared in SDS but not enzymatically degraded was stained with propidium iodide (PI, red-nucleus) and calcofluor white (CW, green—cell wall) to assess the retention of DNA and cellular structure (Fig. 2). Using CLSM the whole, intact tobacco leaf disc was imaged and the z-stacks were assembled into a raw, unprocessed 3D slice reconstruction (Fig. 2B). Optical penetration through the entire leaf thickness (~150 μm) was achieved. Vascular, epidermal, palisade mesophyll and spongy mesophyll cell structure was maintained with all nuclei fixed within the cytosol of the cell. Even long trichome protrusions remained perpendicular to the epidermal surface layer across the entire leaf disc (see supplementary 1 for basal trichome orientation; full trichomes were not imaged). Furthermore the staining of nuclei by PI demonstrated the retention of nuclear DNA. This technique also allowed deep optical penetration into the vascular bundle with nucleus of the sieve element / companion cell (SE/CC) clearly defined without the need for sectioning or epidermal peeling.

Bottom Line: Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration.Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves.Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW, 2308, Australia.

ABSTRACT
Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

No MeSH data available.