Abstract
The small size of ciliary structures that underlies photoreceptor function and inherited ciliopathies requires imaging techniques adapted to visualizing them at the highest possible resolution. In addition to powerful super-resolution imaging modalities, emerging approaches to sample preparation, including expansion microscopy (ExM), can provide a robust route to imaging specific molecules at the nanoscale level in the retina. We describe a protocol for applying ExM to whole retinas in order to achieve nanoscale fluorescence imaging of ciliary markers, including tubulin, CEP290, centrin, and CEP164. The results are consistent with those from other super-resolution fluorescence techniques and reveal new insights into their arrangements with respect to the subcompartments of photoreceptor cilia. This technique is complimentary to other imaging modalities used in retinal imaging, and can be carried out in virtually any laboratory, without the need for expensive specialized equipment.
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Change history
02 September 2023
A correction has been published.
Abbreviations
- BBS:
-
Bardet-Biedl syndrome
- BSA:
-
bovine serum albumin
- CC :
-
connecting cilium
- DAPI:
-
4′,6-diamidino-2-phenylindole
- ExM:
-
expansion microscopy
- IFT:
-
intraflagellar transport
- NGS:
-
normal goat serum
- OCT:
-
optimal cutting temperature
- PBS:
-
phosphate-buffered saline
- PFA:
-
paraformaldehyde
- RT:
-
room temperature
- SDS:
-
sodium dodecyl sulfate
- SIM:
-
structured illumination microscopy
- STORM:
-
stochastic optical reconstruction microscopy
- TEMED:
-
tetramethylethylenediamine
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Acknowledgments
This work was supported by NIH grant R01-EY26545 (TGW), F32-EY027171 (MAR), T32-EY007102 (ARM), and F32-EY031574 (ARM) and a grant from the Knights Templar Eye Foundation (MAR). 3D deconvolution analysis was performed in the West Virginia University Microscope Imaging Facility, which has been supported by the WVU Cancer Institute and NIH grants P20RR016440, P30GM103488, and U54GM104942.
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The authors have declared that no conflicts of interest exist.
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Moye, A.R., Robichaux, M.A., Wensel, T. (2023). Expansion Microscopy of Mouse Photoreceptor Cilia. In: Ash, J.D., Pierce, E., Anderson, R.E., Bowes Rickman, C., Hollyfield, J.G., Grimm, C. (eds) Retinal Degenerative Diseases XIX. Advances in Experimental Medicine and Biology, vol 1415. Springer, Cham. https://doi.org/10.1007/978-3-031-27681-1_58
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DOI: https://doi.org/10.1007/978-3-031-27681-1_58
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