Advertisement

Preservation of Photoreceptor Nanostructure for Electron Tomography Using Transcardiac Perfusion Followed by High-Pressure Freezing and Freeze-Substitution

  • Stefanie VollandEmail author
  • David S. WilliamsEmail author
Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1074)

Abstract

The phototransductive membrane disks of a vertebrate photoreceptor outer segment (OS) are highly susceptible to perturbations during preservation for electron microscopy. To optimize their preservation for nanostructural studies, such as with electron tomography (ET), we developed a protocol, using a combination of chemical and physical fixation approaches, including transcardiac perfusion, high-pressure freezing, and freeze-substitution.

Keywords

Photoreceptor Electron tomography High-pressure freezing Freeze-substitution Disk membrane morphogenesis 

Notes

Acknowledgments

This study was supported by NIH grants R01EY24667, R01EY13408, and P30EY00331. We thank Ivo Atanasov for technical assistance and acknowledge the use of instruments at the Electron Imaging Center for Nanomachines, supported by UCLA and NIH grant S10RR23057.

References

  1. McDonald KL, Auer M (2006) High-pressure freezing, cellular tomography, and structural cell biology. BioTechniques 4:137–143. 139, 141CrossRefGoogle Scholar
  2. Murk JL, Posthuma G, Koster AJ et al (2003) Influence of aldehyde fixationon on the morphology of endosomes and lysosomes: quantitative analysis and electron tomohgraphy. J Microsc 212:81–90CrossRefGoogle Scholar
  3. Sosinsky GE, Crum J, Jones YJ et al (2008) The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J Struct Biol 161:359–371CrossRefGoogle Scholar
  4. Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203:285–294CrossRefGoogle Scholar
  5. Volland S, Louise CH, Kong C et al (2015) Three-dimensional organization of nascent rod outer segment disk membranes. PNAS 112:14870–14875CrossRefGoogle Scholar
  6. Young RW (1967) The renewal of photoreceptor cell outer segments. J Cell Biol 33:61–72CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Ophthalmology, Stein Eye Institute, UCLA School of MedicineLos AngelesUSA
  2. 2.Department of NeurobiologyUCLA School of MedicineLos AngelesUSA
  3. 3.Molecular Biology Institute, UCLA School of MedicineLos AngelesUSA
  4. 4.Brain Research Institute, UCLA School of MedicineLos AngelesUSA

Personalised recommendations