Application of a New High-Pressure Freezing Apparatus

Krisp, Holger; Wichert, Götz von; Seufferlein, T; Walther, Paul

doi:10.1017/s1431927606067006

Cited by 1 publication

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References 2 publications

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“…An exciting prospect in modern microscopy is the ability to correlate results from light microscopy and TEM on the same high‐pressure frozen specimen (Biel et al , 2003; Pelletier et al , 2006; Müller‐Reichert et al , 2007). The ability to preserve both recombinant and synthetic fluorophores through HPF, FS and resin embedment (Biel et al , 2003; Luby‐Phelps et al , 2003; Hardie et al , 2004; Krisp et al , 2006) along with the development of photo‐conversion techniques (Gaietta et al , 2002; Grabenbauer et al , 2005) broadens this potential to augment pre‐existing live cell fluorescence studies with direct ultrastructural correlation. Further extension from 2D to 3D data is available through the use of confocal LM at sub‐micrometre resolution, and TEM tomography at nanometer resolution (Frey et al , 2006).…”

Section: Resultsmentioning

confidence: 99%

Controlled microaspiration for high‐pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

Triffo

Pálsdóttir

McDonald

et al. 2008

Journal of Microscopy

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SummaryHigh-pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of highpressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.

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