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Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

National Institutes of Health

WormGUIDES.org

We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.

Single-shot super-resolution total internal reflection fluorescence microscopy.

Excessive alcohol consumption is associated with neuroinflammation, which likely contributes to alcohol-related pathology. However, positron emission tomography (PET) studies using radioligands for the 18-kDa translocator protein (TSPO), which is considered a biomarker of neuroinflammation, reported decreased binding in alcohol use disorder (AUD) participants compared to controls. In contrast, autoradiographic findings in alcohol exposed rats reported increases in TSPO radioligand binding. To assess if these discrepancies reflected differences between in vitro and in vivo methodologies, we compared in vitro autoradiography (using [ H]PBR28 and [ H]PK11195) with in vivo PET (using [ C]PBR28) in male, Wistar rats exposed to chronic alcohol-vapor (dependent n = 10) and in rats exposed to air-vapor (nondependent n = 10). PET scans were obtained with [ C]PBR28, after which rats were euthanized and the brains were harvested for autoradiography with [ H]PBR28 and [ H]PK11195 (n = 7 dependent and n = 7 nondependent), and binding quantified in hippocampus, thalamus, and parietal cortex. Autoradiography revealed significantly higher binding in alcohol-dependent rats for both radioligands in thalamus and hippocampus (trend level for [ H]PBR28) compared to nondependent rats, and these group differences were stronger for [ H]PK11195 than [ H]PBR28. In contrast, PET measures obtained in the same rats showed no group difference in [ C]PBR28 binding. Our in vitro data are consistent with neuroinflammation associated with chronic alcohol exposure. Failure to observe similar increases in [ C]PBR28 binding in vivo suggests the possibility that a mechanism mediated by chronic alcohol exposure interferes with [ C]PBR28 binding to TSPO in vivo. These data question the sensitivity of PBR28 PET as a methodology to assess neuroinflammation in AUD.

Detecting neuroinflammation in the brain following chronic alcohol exposure in rats: A comparison between in vivo and in vitro TSPO radioligand binding.

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacin as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization.

Min Guo

Section on High Resolution Optical Imaging,
National Institute of Biomedical Imaging and Bioengineering,
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering

National Institutes of Health

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Min Guo

Section on High Resolution Optical Imaging,National Institute of Biomedical Imaging and Bioengineering,Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering

National Institutes of Health

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Section on High Resolution Optical Imaging,
National Institute of Biomedical Imaging and Bioengineering,
Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering