研究目的
Investigating the role of actin reorganization in neurite dystrophy after trauma using high speed structured illumination microscopy and lattice light sheet microscopy.
研究成果
High speed SIM and LLSM were successfully applied to observe reorganization of the actin cytoskeleton in living, traumatized neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide, suggesting dystrophy arose from loss of mechanical tension rather than active reorganization of the actin cytoskeleton. These methods can be used to investigate genetic factors and therapeutic interventions that modulate neurite dystrophy after trauma.
研究不足
The immaturity of hiPSCNs limits the range of questions that can be addressed with this model. The study could not address the influence of jasplakinolide at later times due to signal decline in treated samples.
1:Experimental Design and Method Selection:
The study applied high speed structured illumination microscopy (SIM) and lattice light sheet microscopy (LLSM) to observe the actin cytoskeleton in living, traumatized neurons. Methods for traumatizing neurons before imaging and analyzing SIM images to quantify post-traumatic neurite dystrophy were introduced.
2:Sample Selection and Data Sources:
Human induced pluripotent stem cell-derived neurons (hiPSCNs) were used. Cultures were stained with SiR-Actin for SIM and with DiI and SiR-Actin for LLSM.
3:List of Experimental Equipment and Materials:
Equipment included a structured illumination microscope and a lattice light sheet microscope. Materials included hiPSCNs, SiR-Actin, DiI, jasplakinolide, and other cell culture reagents.
4:Experimental Procedures and Operational Workflow:
Neurites were transected using a glass capillary mounted on a micromanipulator for SIM experiments and a special tool for LLSM experiments. Cultures were imaged before and after trauma.
5:Data Analysis Methods:
A semi-automated process in MATLAB was used to segment and analyze SIM images to quantify neurite dystrophy.
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micromanipulator
Leica
Leica
Transecting neurites for SIM experiments.
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sCMOS camera
Orca Flash 4.0 v2
Hamamatsu
Detecting emissions in SIM and LLSM experiments.
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liquid crystal variable retarder
SWIFT
Meadowlark
Rotating the polarity of the light to match the angle of the pattern in SIM experiments.
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structured illumination microscope
Imaging the actin cytoskeleton at super-resolution resolution in living cells.
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lattice light sheet microscope
Imaging the actin cytoskeleton and the overall shape of the neurite with high spatiotemporal resolution.
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spatial light modulator
BVO AHWP3
Bolder Vision Optik
Producing excitation patterns in SIM experiments.
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stage-top incubator
H301
Okolabs
Maintaining samples at 37 °C and 5% CO2 during SIM experiments.
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detection objective
CFI Apo LWD 25XW
Nikon
Collecting fluorescent emission in LLSM experiments.
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excitation objective
0.65 NA, 3.74-mm WD
Special Optics
Illuminating samples in LLSM experiments.
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diode lasers
MPB Communications
Providing illumination in LLSM experiments.
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acousto-optic tunable filter
AOTF
Controlling laser transmittance in LLSM experiments.
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