Journal of Microscopy, Vol. 230, Pt 2 2008, p. 252

Introduction

Themed Issue on High-Pressure Freezing

“A weak point of the present technique is the delay of several secondsbetween specimen sampling and cryofixation. For example, celldynamics, one of the most important properties of life, is still beyondthe scope of applications, despite these hopeful approaches that havebeen made to freeze suspended living micro organisms directly intheir natural growth medium. Further efforts and improvementsare required to achieve time-resolved cryofixation by high-pressurefreezing.”

Taken from the foreword of the first specialized issue on high-pressure freezing (J. Mic. 212, 2003).

Most of the following articles were presented during aconference on ‘New Developments in High-Pressure Freezing’on 9–11 October 2006, at the Max Planck Institute ofMolecular Cell Biology and Genetics (MPI-CBG), in Dresden,Germany. This was the 2nd International Meeting on High-Pressure Freezing, the first being held in Bad Schandau,Germany in 2002. Since that first meeting the interestin high-pressure freezing (HPF) has increased considerably,due perhaps to the increasing interest in cellular electrontomography and vitreous cryosectioning. Researchers inthese latter areas insist on having the best ultrastructuralpreservation of biological material they can get. Importantly,for cells and tissues larger than 10 μm, the fixation methodof choice is HPF. Manufacturers have also responded to thedemands of the research community by producing three newversions of high-pressure freezers in recent years. Around2003, Martin Wohlend Engineering (Sennwald, Switzerland)produced the Wohlwend HPF Compact 01. In 2005, LeicaMicrosystems (Vienna, Austria) came out with the EM PACT2with Rapid Transfer System, and in 2007 BAL-TEC AG(Liechtenstein) unveiled the HPM 100. The recent Leica andBAL-TEC systems are designed for correlative LM-EM (CLEM)and aim to achieve better time-resolved cryofixation for suchcorrelative studies.

The papers in this themed issue reflect different areasof interest using HPF. Triffo et al. report on the use of amicromanipulator and cellulose microcapillary tubing as ameans of protecting and handling delicate or sparse samples

for HPF. Another paper by Hagen & Grunewald shows howdextran microcarriers can be used for HPF of adherentcells for subsequent vitreous cryosectioning. Two papersdeal with how to process samples following HPF: Buser &Walther explore how and why water, which is added tothe freeze-substitution cocktail, enhances the visualizationof cell membranes. Morphew et al. demonstrate that silverenhancement of ultrasmall gold particles can be achieved inorganic solvents during freeze substitution.

Significantly, four of the papers included here use some kindof correlative light microscopy (LM) with HPF. We are greatlyencouraged by this because we believe that LM in combinationwith HPF and electron microscopy (EM) have many importantapplications to today’s biology. A correlative approach takesadvantage of the huge database of LM observations in livingcells, and combines the contextual information from LMwith the resolution of EM. It allows us to ‘fix’ a dynamicprocess at a known point or to locate a rare event orstructure. Moreover, it dramatically increases EM sample sizeand throughput for certain types of studies. Along these lines,Paul Verkade describes the development and some applicationsof the Rapid Transfer System for the Leica EM PACT2 high-pressure freezer. Bouchet-Marquis & Grabenbauer correlateLM immunofluorescence of Golgi apparatus with the EManalysis of vitreous cryosections. Vanhecke et al. compareLM images of Walker carcinosarcoma cells with similarregions seen in the EM of thin sections to identify a neworganelle. Finally, Muller-Reichert et al. use LM and time-resolved cryofixation by HPF to achieve tomographic analysesof mitotic spindle components in early Caenorhabditis elegansembryos.

It has taken a long time for HPF, generally available inthe 1980s, to be currently appreciated. With the adventof new equipment, and increasing interest in cellulartomography, vitreous sectioning techniques, and correlativeLM-EM methods, the future of HPF as a fundamental tool instructural cell biological research is secure.

KENT MCDONAL D (Berkeley)THOMAS MUL L ER-REICHERT (Dresden)

C© 2008 The AuthorJournal compilation C© 2008 The Royal Microscopical Society