Why Is The Glow Plug Light Flashing In My VW & What Does It Indicate
NHI 1.pptx
1. Structural and molecular interrogation
of intact biological systems
Nature. 2013 May 16; 497(7449): 332–337. doi:10.1038/nature12107
NGUYEN THI NHI
Master’s Student
Department of Physiology
3. • Automatic sectioning
• Microscopy
• Imaging methods
Map structure
NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS 3
4. 4
BigBrain
An Ultrahigh-Resolution3D
Human Brain Model
NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS
- 65-year-old male
- Cutting brain (coronally - 20µm)
- Histological stain
- 3D-reconstructed image
6. NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS 6
• Brain of 3-5-week-old mouse
• Surface of a whole mouse brain
reconstructed from 550 optical
sections
Ultramicroscopy: three-
dimensional visualization of
neuronal networks in the
whole mouse brain
8. NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS 8
Optically transparent and
macromolecule-permeable construct
& Preserving native molecular
information and structure
CLARITY
9. NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS
9
acrylamid
e
formaldehyd
e
biomolecule-conjugated
monomer
hybrid
construct
CLARITY
14. The Power of PowerPoint | thepopp.com 14
Molecular
phenotyping
Figure 4
15. 15
• Imaging of local circuit wiring cellular relationships,
subcellular structures, protein complexes, nucleic acids,
…
• CLARITY also enables intact-tissue immuno-
histochemistry with multiple rounds, and antibody
labelling throughout the intact adult mouse brain.
MOUSE
NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS
16. NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS 16
HUMAN
Figure 5
Figure 5
17. 17
• CLARITY enables fine structural analysis of
human samples at subcellular level.
• Intact and accessible form suitable for
probing structural and molecular
underpinnings of physiological function and
disease.
HUMAN
NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS
18. TAKE-HOME MESSAGE
18
Transforms intact tissue into a nanoporous hydrogel-hybridized for
Optically transparent and macromolecule-permeable
construct.
Preserves native molecular information and structure.
NUCLEIC ACID AND GENE BIOCHEMISTRY CLASS
Provides access to support integrative understanding of
large-scale intact biological systems.
CLARIT
Y
Assess, approach, means, technique
Scientist explore/ discover
How did they do that
o create the brain model, we used a large-scale microtome to cut a complete paraffin-embedded brain (65-year-old male) coronally(Fig. 1), and we then acquired 7400 sections at20-mm thickness and stained them for cell bodies(14). Histological sections were digitized, result-ing in images of maximally 13,000 by 11,000 pixels(10-by-10–mm pixel size)..(A) Photographs of the fixed brain. Lateral left(top), lateral right (middle), and dorsal (bottom) views. (B) Magnetic resonance image (coronal view)and (C) 3D-reconstructed MRI volume of the fixed brain. (D) Histological sectioning. (E) Block face image of a section (pseudo colored) resting on the mounting grid that served for alignment of theblock face images. (F) Series of blockface images. (G)Cellbody–stained histological sections with the region of interest denoted by a red box. This area is shown with higher magnification in (H). (I)Series of histological images, which were 3D-reconstructed using the blockface images (F) and the MRI (C).21 JUNE 2013 VOL 340SCIENCEwww.sciencemag.org1472REPORTS
Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now.
Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution
, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains.
The advantage of this method is that the sample is preserved and can be re-imaged, if needed. But no matter how precisely it is done, cutting the sample inevitably results in distortions that make it difficult to align the images
We modified ‘ultramicroscopy’ by combining it with a special procedure to clear tissue.
We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution
and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.
Whereas an alternative technique, optical projection tomography, is inherently limited to a resolution of about 20 microm, our technique allows resolution in the sub micrometer range for small objects (<2 mm). With suitable lenses it should be possible to reach this resolution throughout whole embryos.
Não lớn tuổi, lipid nhiều ?
Our 3D reconstructions are presently limited by computer power, as our graphics processors can not handle more than 600 optical slices with high resolution. Mouse brains older than 2 weeks cannot be imaged completely, as heavily myelinated structures such as thalamus and brain stem do not become transparent
A thin polar membrane made of two layers of lipid molecules.
They took note of the fact that packed lipid bilayers are implicated in making tissue poorly accessible, reconstruction—both to molecular probes and to photons—by creating diffusion-barrier properties relevant
to chemical penetration, as well as light-scattering properties at the lipid–aqueous interface
If lipid bilayers could be removed nondestructively, light and macromolecules might penetrate deep into the tissue, allowing threedimensional imaging and immunohistological analysis without disassembly
/əˈkrɪl.ə.maɪd/ + /fɔːˈmæl.də.haɪd/
In this step, formaldehyde not only crosslinks the tissue, but also covalently links the hydrogel monomers to biomolecules including proteins, nucleic acids and small molecules.
hydrogel–tissue hybridization physically supports tissue structure and chemically incorporates biomolecules into the hydrogel mesh
Electric fields applied across the sample in ionic detergent actively transport micelles into, and lipids out of, the tissue, leaving finestructure and crosslinked biomolecules in place. The ETC chamber is depicted in the boxed region
3-month-old line-H mouse
the intact brain (including heavily myelinated white matter, thalamus and brainstem) becomes uniformly transparent full CLARITY processing enabled imaging of the entire intact mouse brain at cellular resolution even using single-photon microscopy (d-f)
Single-photon microscopy
Figure 2f shows a volume of unsectioned mouse brain with visualization through the cortex, hippocampus and thalamus CLARITY processing enabled imaging of the entire intact mouse brain at cellular resolution even using single-photon microscopy CLARITY thus allows rapid high-resolution optical access to dense intact tissue and, if needed, subsequent electron microscopy analysis. Optical sections from f showing negligible resolution loss even at ~3,400-µm deep:
fluorescent proteins tested, including green, yellow and red fluorescent proteins (GFP, YFP and RFP, respectively), were robust to ionic detergent extraction To quantify molecular preservation associated with tissue–hydrogel fusion, we compared protein loss in clarified mouse brain to loss from conventional methods only ~8% protein loss was seen, indicating that chemical tethering of biomolecules into hydrogel mesh can enhance the preservation of molecular tissue components. increases tissue permeability by replacing lipid bilayers with nanoporous hydrogel, enabled rapid diffusion of molecular probes deep into intact tissue, and therefore allows access to preserved biomolecules without sectioning
a. CLARITY may preserve native antigens with unusual completeness owing to the hydrogel-hybridization process. To quantify molecular preservation associated with tissue–hydrogel fusion, we compared protein loss in clarified mouse brain to loss from conventional methods.
However, when hydrogelhybridized tissue was cleared with the stringent 4% SDS solution of CLARITY, only ~8% protein loss was seen, indicating that chemical tethering of biomolecules into hydrogel mesh can enhance the preservation of molecular tissue components
b. which increases tissue permeability by replacing lipid bilayers with nanoporous hydrogel, enabled rapid diffusion of molecular probes deep into intact tissue, and therefore allows access to preserved biomolecules without sectioning. In a 1-mm-thick clarified coronal block of mouse brain, uniformly antibody stained over 3 days
d. Mander’s overlap coefficient was used to quantify the overlap between corresponding fluorescent signals derived from Cx36 and cell organelles in co-transfected cells. Co-localization was accepted at a Mander’s coefficient > 0.5; this threshold indicated that the occurrence of true co-localization surpassed the probability of chance Quantitative co-localization analysis revealed that eYFP fluorescence and anti-GFP staining overlapped throughout the block
enabled rapid diffusion of molecular probes deep into intact tissue, and therefore allows access to preserved
To investigate axonal projections of the tyrosine hydroxylase neurons further, we clarified, tyrosine-hydroxylase-stained and imaged 1-mm-thick coronal blocks of mouse brain using a high numerical aperture objective (0.95) - is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance
Throughout the tissue volume we could unequivocally identify individual paired preand postsynaptic puncta, molecularly defining identity and position of putative excitatory synapses
e. Right, enlarged images of boxed regions on left. Individual synaptic puncta resolved throughout depth f.g. Average immunofluorescence cross-section of PSD-95 (Postsynaptic density protein 95) puncta. . The full-width at half-maximum of the point-spread function was uniform throughout the block, indicating that loss of resolution is negligible even near the diffraction limit of conventional light microscopy
demonstrating another feature of the technique: removal of lipid membranes ensures that tissue refractive index remains nearly constant throughout large volumes, allowing high-resolution imaging h.i. We also found that the CLARITY hydrogel-conjugation process preserves small molecules such as the neurotransmitter GABA (γ-aminobutyric acid, Fig. 3h) and (for in situ hybridization) messenger RNAs (Fig. 3i), paving the way for multimodal combinatorial labelling within unsectioned tissue
CLARITY was found to enable multi-round molecular phenotyping; the stable framework allowed effective removal of antibodies without fine structural damage or degraded antigenicity. We performed three consecutive rounds of staining in 1-mm-thick coronal blocks from a Thy1–eYFP H-line mouse brain, observing effective antibody removal and preserved eYFP-positive
neuronal morphology as well as re-staining capability.
Although further quantification would be required to fully map the extent to which CLARITY secures molecular information, this result
shows that elution in clarified tissue largely preserves integrity of tissue structure, cellular architecture, fluorescence signals (Fig. 4d–f) and 4′,6-diamidino-2-phenylindole (DAPI) DNA staining (Fig. 4g).
Moreover, repeated tyrosine hydroxylase staining in the first and third rounds maintained signal pattern and intensity, confirming that antigenicity is retained throughout multiple rounds of staining and elution. a, First round. Rendering of 1-mm-thick Thy1–eYFP block immunostained for tyrosine hydroxylase in non-sectioned form.
b, Antibodies eluted from block in a (4% SDS, 60 °C for 0.5 days). Tyrosine hydroxylase signal was removed and eYFP fluorescence retained c, Second round. Three-dimensional rendering of same block now immunostained for parvalbumin (red), glial fribrillary acidic protein (GFAP) (blue) and DAPI (white) d-f. Maximum projections of 100 µm volume of yellow-boxed regions in a, b and c, respectively. eYFP-positive neurons preserved. cp, cerebral peduncle; SNR, substantia nigra. Scale bar, 100 µm.
g, Optical section of white/ dotted-box region in c showing DAPI. CA, cornu ammonis; DG, dentate gyrus. Scale bar, 100 µm. h, i, Tyrosine hydroxylase channel of white box regions in a (h) and j (i). Tyrosine hydroxylase antigenicity preserved through multiple elutions. Scale bar, 100 µm.
j, Third round. Block in a–c immunostained for tyrosine hydroxylase (red) and choline acetyltransferase (ChAT) (blue) k, Three-dimensional view of hippocampus in c showing eYFP-expressing neurons (green), parvalbumin-positive neurons (red) and GFAP (blue). Alv, alveus. Scale bar, 200 µm
Using mouse brains,
They found that CLARITY was suitable for such long-banked human brain, allowing immunohistological visualization and identification of neurons and projections over large volumes (a-g).
In 0.5-mm-thick blocks of frontal lobe from an autistic patient, stored in formalin for >6 years, we were able to stain for axons with neurofilament protein and myelin basic protein,
and trace individual fibres (e). we found that many parvalbum-inpositive interneurons in this human sample, particularly in deep layers, showed isoneuronal and heteroneuronal dendritic bridges (g-m)
. In addition, by staining for parvalbumin it was possible to visualize the distribution of parvalbumin-positive neurons in the neocortex over large volumes (6.7 × 4.7 × 0.5 mm),
and trace individual parvalbumin-labelled processes Human BA10 500-µm-thick intact blocks clarified (1 day) and immunostained (3 days) (×25 water-immersion objective).
a, Optical section: myelin basic protein (MBP) and parvalbumin staining. White arrowheads indicate membrane-localized myelin basic protein around parvalbumin-positive projections. Scale bar, 10 µm. b, Tyrosine hydroxylase and parvalbumin staining.
c, Optical section: neurofilament (NP) and GFAP. Scale bar, 20 µm. d, Somatostatin and parvalbumin staining Rendering of neurofilament-positive axonal fibres. Red, traced axon across volume. Scale bar, 500 µm. Inset: boxed region. f, Visualization of parvalbumin-positive neurons in the neocortex of autism case; layers identified as described in ref.
g, Yellowboxed region in f showing parvalbumin-positive cell bodies and fibres in layers 4, 5 and 6. Three representative parvalbumin-positive interneurons in layer 6 with ladder-shaped hetero- or iso-neuronal connections were traced (green, purple, blue). Scale bar, 100 µm h, Three-dimensional rendering of abnormal neurons in g; yellow arrowheads (1, 2) indicate ladder-shaped structures shown below in i and k.. i, Zoomed-in maximum projection of 8 µm volume showing morphology of ladder-shaped structure formed by neurites from a single neuron.
j, Tracing of structure in
k, Maximum projection of 18 µm volume showing ladder-shaped structure formed by neurites from two different neurons.
l, Tracing of structure in k. m, Iso- and hetero-neuronal dendritic bridges per neuron. Neurons selected randomly and traced in software (Methods);
dendritic bridges were manually counted. **P<0.05; error bars denote s.e.m. n=6 neurons for both
superficial and deep layers of autism case and n=4 neurons for both superficial and deep layers of control case.
n, Threedimensional reconstruction of a neuron in layer 2 (superficial) of the autism case. Typical avoidance of iso-dendritic contact was observed
