Rapid 3D-STORM imaging of diverse molecular targets in tissue

Albrecht, Nicholas E.; Jiang, Danye; Akhanov, Viktor; Hobson, Robert J.; Speer, Colenso M.; Robichaux, Michael A.; Samuel, Melanie A.

doi:10.1016/j.crmeth.2022.100253

Cited by 4 publications

(2 citation statements)

References 58 publications

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“…For STORM, thin retina resin sections were collected in 35 mm glass-bottom dishes (MatTek Life Sciences, Cat# P35G-1.5-14-C), chemically etched in a mild sodium ethoxide solution (approximately 1% diluted in pure ethanol for 0.5 to 1.5 h), as previously described [ 57 ]. Prior to STORM imaging, etched sections were mounted in a STORM imaging buffer adapted from [ 58 ]: 50 mM Tris (pH 8.0), 10 mM NaCl, 10 mM sodium sulfite, 10% glucose, 40 mM cysteamine hydrochloride (MEA, Chem Impex/VWR, Cat# 102574–806), 143 mM BME, and 1 mM cyclooctatetraene (Sigma Cat# 138924), under a #1.5 glass coverslip that was sealed with quick-set epoxy resin (Devcon).…”

Section: Methodsmentioning

confidence: 99%

“…Both structured illumination microscopy (SIM), and stochastic optical reconstruction microscopy (STORM), a single-molecule localization microscopy, were previously used to localize ciliary proteins to the nanometer-scale subcompartments of the CC in mouse rods [ 24 , 57 ]. More recently, an alternative 3D STORM mode, named rapid imaging of tissues at the nanoscale STORM (RAIN-STORM), was developed to localize proteins in rod photoreceptor presynaptic terminals and postsynaptic dendrites within the mouse OPL [ 58 ]. To enable super-resolution localization of Rho in the mouse IS, we applied techniques to reliably immunolabel Rho in the IS of mouse retina, including an OS peeling approach, nanobody targeting of Rho-GFP in Rho-GFP/+ knock-in retinas, and surface labeling for SIM and STORM followed by a quantitative localization analysis.…”

Section: Introductionmentioning

confidence: 99%

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Super-resolution mapping in rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway

Haggerty,

Eshelman,

Sexton

et al. 2024

PLoS Biol

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Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient phototransduction and vision. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane, at the surface, in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Super-resolution mapping in rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway

Haggerty,

Eshelman,

Sexton

et al. 2024

PLoS Biol

Self Cite

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Neuronal signal-regulatory protein alpha drives microglial phagocytosis by limiting microglial interaction with CD47 in the retina

et al. 2022

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Mapping rhodopsin trafficking in rod photoreceptors with quantitative super-resolution microscopy

Haggerty

Eshelman

Sexton

et al. 2023

Preprint

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Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient long-term phototransduction. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors.SUMMARYPhotoreceptor cells of the retina are maintained through a complex protein trafficking network. This study applies quantitative super-resolution microscopy to uncover localization details about the trafficking of the essential visual pigment rhodopsin in the inner segment region of rod photoreceptors.

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Rapid 3D-STORM imaging of diverse molecular targets in tissue

Cited by 4 publications

References 58 publications

Super-resolution mapping in rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway

Super-resolution mapping in rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway

Neuronal signal-regulatory protein alpha drives microglial phagocytosis by limiting microglial interaction with CD47 in the retina

Mapping rhodopsin trafficking in rod photoreceptors with quantitative super-resolution microscopy

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