Sapphire disk quick-freeze "chicken & eggs" protocol

Heuserlab sapphire-disk quick-freeze “chicken ‘n eggs” protocol:

To actually witness the interactions of nanoscale physical entities with living cells at high resolution in the electron microscope, and to do so from the point of view of a substrate on which the nanoscale entities have been adsorbed, our lab's approach is basically the following:

  1. Attach the experimental entities to the substratum, either by simple adsorption or by 'spinoculation” (centrifuging the entities down onto the substrate).
  2. Add cells on top and let them interact with the nanoscale entities... that is, to physically interact with them, to sit on the material like chickens sitting on their eggs.

This novel approach offers several specific advantages:

  1. The cells lie flat on the substrate, and this provides broad views of the bottoms of the cells and  their  interactions with the mesoscale entities.
  2. Such 'sandwiching' of the mesoscale material between substrate and cells creates an ideal close-approach between the two, thus achieving maximal substrate concentration and maximal opportunity for interaction with the cells.

How we actually go about seeing the bottoms cells and their interactions with nanoscale substances, and how we see this from the substrate's vantage point is quite simply by:

  1. “Slam”-freeze the 'sandwich' (cells/nanoscale substance/and substrate) from beneath, by applying the coolant to the backside of the substrate.  (This would not work if the substrate were made of glass; but we have recently discovered that it works extremely well if the substrate is made of natural sapphire, a great conductor of temperature.) (See below for historical uses of sapphire in freezing, and current sources for purchasing sapphire.)
  2. Turn the sample over, pop off the substrate off from the sample while it is remains frozen, and finally replicate or otherwise image in the transmission or scanning EM the exposed “underbellies” of the cells.

Whatever nanooscale material that was on the substrate and stuck to the cells will thereby be beautifully represented on the cells' underbellies. 

Finally, once the behavior of the cell vis-a-vis the nanomaterial-'doped' substrate is understood, the whole EM experiment can be reversed, “tipped over on its head” so-to-speak, and the cells can be “unroofed” while attached to the same substrate.  In this manner, the inside surfaces of the ventral-most attached membranes can be visualized in exactly the same way as was done above for the outside surfaces. (See our protocols for “unroofing” cells, elsewhere on this site.)

Thereby, we are able to readily compare both sides of any differentiated cell membrane, and both at the electron microscopic level, and we can relate whatever 'effects' we witness on the outside of the cell to whatever 'causes' we find to be operative on the inside of the cell (namely, any cytoskeletal and other cytoplasmic activities and structural changes that we observe).

Sources of sapphire disks for the above protocol:

Many years ago, at the recommendation of Thierry Soldati**, we ordered 'sapphire cover slips' from these folks:

Groh + Ripp
Tiefensteiner Strasse 322 a
55743 Idar-Oberstein
GERMANY

Tel.: +49 (0) 6781 93 50 0
Fax: +49 (0) 6781 93 50 50

Attn: Stefanie Ripp or Sandra Ripp-Brunk, (or currently: Christina Kaba)

We used to get from this company:
6mm dia disks 0.05mm thick, at DM 10 each, and
3mm dia disks 0.15mm thick, at DM 10 each.

Currently, they offer the following:
“Sapphire cover slips 0,05mm thick, both sides polished”
Sizes: 3mm ø or 6mm ø
Minimum order: 100 pieces @ € 12,00 p.pc (for a total of €1200)

More recently, Toyoshi Fugimoto <tfujimot@med.nagoya-u.ac.jp>
bought excellent sapphire disks from:

Martin Wohlwend GmbH
CH-9466
Sennwal
Switzerland

Telefon: 081/7571924
Telefax: 081/7572243

martin-wohlwend@bluewin.ch

These were called “Specimen holders for HPFM” and were described as:
“Sapphire disks, dia. 3x0.05mm, article no. 405”
Price per unit in CHF: 4.50, min. order quantity 100 (total in CHF: 450.00)

On the other hand, Dr. Jiro Usukura, a colleague of Dr Fugimoto,

<usukuraj@esi.nagoya-u.ac.jp>

apparently purchases sapphire disks from another outfit:

Rudolf Brugger S.A..
via Decio Bacilieri 24
Minusio,
CH-6648
Switzerland

Tel: +41 91 7435413
Fax: +41 91 7435460

E-mail: info@rudolfbrugger.com
contact: gianella@rudolfbrugger.com

http://www.rudolfbrugger.com

(I don't know what these folks offer, or what they charge)

Apparently, this source came from a letter Pat Echlin wrote some years ago:

Echlin, P (2003)
Letter to the Editor
J. Microscopy 212: 101

I have read with great interest the recent paper in the Journal of Microscopy, vol. 209 pp. 76-80, 2003 by Reipert et al. on the use of sapphire discs for impact cooling.

The thermal properties of sapphire at very low temperatures were first proposed by Meisner and Hagins in 1978 (Biophys. J. 21, 149a) and the idea expanded by Bill Bald in his book Quantitative Cryofixation, Adam Hilger 1987.

More information can be found in the book by Tony Robards and Uve Sleyter Low temperature methods in biological electron microscopy, Vol. 10 Practical Electron Microscopy. Ed. Audrey Glauert, Elsevier 1985 and in my own book Low Temperature Microscopy and Analysis, Plenum 1992.

Without going into great technical detail, sapphire at 20 K has a thermal conductivity of 15 700 J m_1 s _1 K_1 compared to 10 500 for copper, 1500 for gold and 5000 for silver. At liquid nitrogen temperature (77 K) the comparable figures are 960, 570, 252 and 471.

Now at long last, a company in Switzerland (http://www.rudolfbrugger.com) has produced some small thin sapphire discs which should allow us, relatively easily, to quench cool specimens without using the rather expensive, but nevertheless effective, high pressure cooling equipment.

The sapphire discs may be purchased from Rudolf Brugger SA (contact gianella@rudolfbrugger.com for a quotation). The Reipert et al. paper only shows some TEM images of freeze substituted specimens and it remains to be seen whether sapphire discs will be useful for studying frozen hydrated samples in the SEM and by X-ray microanalysis.

History of the use of sapphire in freezing is as follows:

Neuhaus EM, Horstmann H, Almers W, Maniak M, Soldati T. (1998). Ethane-freezing/methanol-fixation of cell monolayers: a procedure for improved preservation of structure and antigenicity for light and electron microscopies. J Struct Biol. 121:326-342.

Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Heidelberg, Germany.

In order to dissect at the ultrastructural level the morphology of highly dynamic processes such as cell motility, membrane trafficking events, and organelle movements, it is necessary to fix/stop time-dependent events in the millisecond range. Ideally, immunoelectron microscopical labeling experiments require the availability of high-affinity antibodies and accessibility to all compartments of the cell. The biggest challenge is to define an optimum between significant preservation of the antigenicity in the fixed material without compromising the intactness of fine structures. Here, we present a procedure which offers an opportunity to unify preparation of cell monolayers for immunocytochemistry in fluorescence and electron microscopy. This novel strategy combines a rapid ethane-freezing technique with a low temperature methanol-fixation treatment (EFMF) and completely avoids chemical fixatives. It preserves the position and delicate shape of cells and organelles and leads to improved accessibility of the intracellular antigens and to high antigenicity preservation. We illustrate the establishment of this procedure using Dictyostelium discoideum, a powerful model organism to study molecular mechanisms of membrane trafficking and cytoskeleton.

Reipert S, Fischer I, Wiche G.(2003). Cryofixation of epithelial cells grown on sapphire coverslips by impact freezing. J Microsc. 209:76-80.

Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Vienna Biocenter, 1030 Vienna, Austria. ultsr@abc.univie.ac.at

ABSTRACT: Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire-attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal-mirror impact freezing. The potential of the Leica EM-CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze-substitution (SD-FS unit) has been exploited. Subsequently, the SD-FS unit has been tested for its use in methanol freeze-substitution and low temperature embedding for immunoelectron microscopy. The structural preservation of Lowicryl HM20-embedded cells has been assessed as being free of damage by large ice crystals.

RESULTS: “As a result of HPF, we obtained cryoimmobilized cells on sapphire discs, which were tightly bound to the sample holder. Because separation of the sapphire discs from the flat sample holders at cryo-temperatures was not practicable, we performed freeze-substitution while the samples remained in their holders. Under these conditions, the cryoprotectant, which filled the cavity of the sample holder, had a profound effect on the samples. The use of 1-hexadecene prevented effective freeze-substitution, as suggested previously (Hohenberg, H., Mannweiler, K. & Müller, M. (1994) High-pressure freezing of cell suspensions in cellulose capillary tubes. J. Microsc . 175, 34- 43).

Reipert S, Fischer I, Wiche G. (2004). High-pressure freezing of epithelial cells on sapphire coverslips.
J Microsc. 213:81-85.

Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Vienna Biocenter, 1030 Vienna, Austria. ulstr@abc.univie.ac.at

ABSTRACT: Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage-free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing. In a novel approach, we tested the suitability of sapphire discs as cell substrata for high-pressure freezing. Frozen samples on sapphire were subjected to freeze-substitution while in the same flat sample holders as used for high-pressure freezing. We obtained cells that displayed an excellent preservation of fine structure. Because sapphire is a tissue culture substratum suitable for light microscopy, its use in combination with high-pressure freezing could become a powerful tool in correlative studies of cell dynamics at light and electron microscopic levels.

Reipert, S., Fischer, I. & Wiche, G. (2004). Corrigendum. J. Microscopy 215: 313

Some important references were overlooked in our recent contributions covering the application of sapphire coverslip cell cultures to impact freezing and high pressure freezing:

From a historical perspective, HPF of coverslip cell cultures started as early as the late 1980s, when Martin Müller and colleagues (EM-Laboratory, Swiss Federal Institute of Technology/ETH-Zürich) introduced cells grown on 3 mm sapphire coverslips to HPF with an HPM 010 apparatus (from BAL-TEC, Balzers, Lichtenstein).

The following additional literature contains data generated by this technique:

Biomaging, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom

We describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed 'face to face' without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at -90 degrees C for 60 min in acetone containing 2% uranyl acetate, followed by warming to -50 degrees C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.

Hawes PC, Netherton CL, Wileman TE, Monaghan P. (2008). The envelope of intracellular African swine fever virus is composed of a single lipid bilayer. J Virol. 2008 Jun 11 (in press).

Biomaging, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom of Great Britain and Northern Ireland; Vaccinology Group, Division of Immunology, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom of Great Britain and Northern Ireland; Infection and Immunity, School of Medicine, Institute of Health, University of East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom of Great Britain and Northern Ireland.

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryo-sectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV infected cells for EM using chemical fixation, cryo-sectioning and high pressure freezing. The appearance of the intracellular viral envelope was determined and compared to mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high pressure freezing and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryo-sectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different to the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASF viral envelope is composed of a single lipid bilayer.