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Reconstruction
Display image description
Mosaic reconstruction imaged using spinning disk confocal microscopy of a flat mount of retina showing retinal ganglion cells retrogradely labeled via fluorogold injection into the superior colliculus in an adult equivalent wild type mouse (Nf1 flox/flox on C57BL/6 background). Details may be found in Kim et al., Neuroscience. 2010 Sep 29;170(1):178-88.
Full resolution image description
Assembled mosaic in tiff format of retinal ganglion cells retrogradely labeled with fluorogold from a wild type equivalent mouse : 7732_WT-FF.tif.
File format
TIFF
Volume scale
0.41, 0.41, 0.5

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:7732*  Cite 
Project: P2054
Project name
Characterization of optic gliomas from neurofibromatosis-1 (Nf1) genetically-engineered mice
Description
Use of large scale imaging of the optic nerve and chiasm to characterize development of optic gliomas from neurofibromatosis-1 (Nf1) genetically-engineered mice
Funding agency
NCI and NCRR
Leader(s)
Keun-Young Kim
Collaborator(s)
David Gutmann
Eric Bushong
Mark Ellisman
Da-Yong Lee
Start date
09-01-2009
End date
unspecified
 
Experiment
Experiment ID
7723
Title
Large scale mosaic imaging with retrograde labeling of retinal ganglion cells
Purpose
The purpose of this study was to investigate the progression of changes in retinal ganglion cells and optic nerve glia in neurofibromatosis-1 (NF1) genetically-engineered mice with optic glioma. RGC were counted in whole retina preparations using high-resolution, mosaic confocal microscopy following their delineation by retrograde FluoroGold labeling. Then, we found reduced RGC numbers in Nf1+/┬┐GFAPCKO mice, supporting a model in which the combination of optic nerve Nf1 heterozygosity and glial cell Nf1 loss results in disrupted axonal-glial relationships, subsequently culminating in the degeneration of optic nerve axons and loss of their parent RGC neurons.
Experimenter(s)
Keun-Young Kim
Microscopy product
Microscopy product ID
7732
Instrument
Olympus DSU Confocal Microscope
Microscopy type
Spinning Disc Confocal Microscopy
Product type
MOSAIC
Image basename
WT-FF
Subject
Species
mouse
Scientific name
Mus musculus
Strain
C57BL/6 Nf1 flox/flox
Group by
Injection of tracer
Treatment
Both mutant and wild-type mice were injected with fluorogold tracer bilaterally into the superior colliculi. Please refer to the subject description for which images belong to which type of animal. Retrograde Labeling of Retinal Ganglion Cells One week prior to euthanasia, FluoroGold (1 ul/injection; Fluorochrome Inc., Englewood, CO) diluted in saline was microinjected bilaterally into the superior colliculi of anesthetized mice with a mixture of ketamine (Fort Dodge Animal Health) and xylazine (Vedeco Inc.) in a stereotactic apparatus, as previously described (Kim et al., 2004). Fluoro-Gold is taken up by the axon terminals of RGC neurons and transported retrogradely to the cell bodies in the retina (Selles-Navarro et al., 1996). The FluoroGold in the RGC neurons persists for at least 3 weeks without significant fading or leakage (Dong et al., 1996). Mosaic image arrays from retinal flat-mounts (n=6 retinal flat-mounts/mice/group) were captured under an Olympus spinning disk confocal microscope (Olympus America Inc., Center Valley, PA) equipped with a high-precision closed loop XY stage and closed loop Z control with commercial mosaic acquisition software from MicroBrightField (MBF Bioscience Inc., Williston, VT) modified by us (Chow et al., 2006, Price et al., 2006). The microscope was equipped with a high-resolution CCD camera for high-speed mosaic acquisition. Images were stored in Photoshop files (Adobe Systems Inc., San Jose, CA).
Age
9 months
Age class
adult
Tissue section
Anatomical location
eye
Thickness
160 µm
Specimen description
Organ
eye
System
central nervous system
Structure
cell body
Tissue
retina
Cell type
retinal ganglion cell
Imaging parameters
Type
Light microscopy product
Immersion medium
oil
Mounting medium
gelvatol
Lens
Olympus 20x dry
Lens magnification
x
Numerical aperture
1.40
Notes
christine
Specimen preparation
Protocol used
Retrograde labeling of retinal ganglion cellsOne week prior to euthanasia, FluoroGold (1 l/injection; Fluorochrome Inc., Englewood, CO, USA) diluted in saline was microinjected bilaterally into the superior colliculi of anesthetized mice with a mixture of ketamine (Fort Dodge Animal Health) and xylazine (Vedeco Inc.) in a stereotactic apparatus, as previously described. Fluoro-Gold is taken up by the axon terminals of RGC neurons and transported retrogradely to the cell bodies in the retina. The FluoroGold in the RGC neurons persists for at least 3 weeks without significant fading or leakage. Mosaic image arrays from retinal flat-mounts (n=6 retinal flat-mounts/mice/group) were captured under an Olympus spinning disk confocal microscope (Olympus America Inc., Center Valley, PA, USA) equipped with a high-precision closed loop XY stage and closed loop Z control with commercial mosaic acquisition software from MicroBrightField (MBF Bioscience Inc., Williston, VT, USA) modified by us. The microscope was equipped with a high-resolution CCD camera for high-speed mosaic acquisition. Images were stored in Photoshop files (Adobe Systems Inc., San Jose, CA, USA).
Imaging product type
Type
Mosaic
Description
FG labled retinal flatmount