Cryofixation of monolayer cell cultures for freeze‐fracturing without chemical pre‐treatments

Abstract

A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique. 3 mm large discs are punched out and sandwiched with Cu-or Au-object holders of little mass; a 15 pm spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5" , , glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.

Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations

“…Freeze-fracture is a popular SIMS sample preparation method which cryogenically preserves biological samples. 19,22,[28][29][30] Freezefracture is an attractive protocol because it has been shown to maintain the native distribution of molecules in liposomes and cells. 16,[19][20][21][22][23]30 However, freeze-fracture involves many experimental variables, such as the temperature and pressure conditions during the fracture, 30 the portion of the cell exposed to the sample-vacuum interface, 21 and the thickness of ice remaining on the sample substrate, that contribute to experiment-to-experiment variations in secondary ion yield and SIMS images.…”

Mass Spectrometric Imaging of Highly Curved Membranes During Tetrahymena Mating

“…TOF-SIMS has been applied to elemental imaging of the distribution of cancer therapy drugs in single cells (14) as well as molecular imaging of tissue (15) (16,21,22) and directly transferred into the analysis chamber. After TOF-SIMS imaging, the samples were further analyzed by scanning ion microscopy and brightfield microscopy to ensure that no substantial sample damage occurred during sample preparation and SIMS analysis.…”

“…We incubated conjugating Tetrahymena for 4 hours and then placed a small aliquot of the cell-containing solution between two pieces of silicon for cryogenic freezing. The frozen sample was then freeze-fractured under ultrahigh vacuum (16,21,22) and directly transferred into the analysis chamber. After TOF-SIMS imaging, the samples were further analyzed by scanning ion microscopy and brightfield microscopy to ensure that no substantial sample damage occurred during sample preparation and SIMS analysis.…”

The Microstructure of Dilute Clay and Humic Acid Suspensions Revealed by Freeze-Fracture Electron Microscopy: Discussion

“…Secondly, in our re-interpretation of their transmission electron microscopic (TEM) images, we identify the effects of cryo-artifacts. In order to elaborate on this point, we shall discuss their Figures 1-3 by comparing them with results obtained by us (l) with high-rate cryofixation-techniques (Bachmann and Schmitt, 1971;Moor et al, 1976;Miiller et al, 1980;Pscheid et al, 1981) and (2) with conventional freezeetch techniques similar to the ones used by Gu and Doner (1992).…”

“…Cooling rates of this order can be achieved with sprayfreezing (Bachmann and Schmitt, 1971) or sandwichjet-freezing (Moor et al, 1976;Mfiller et al, 1980;Pscheid et al, 1981), which are based on the principle Copyright 9 1992, The Clay Minerals Society of dividing the fluid to be frozen into small enough "parcels." These techniques have been applied successfully to clay-colloidal systems (Vali and Bachmann, 1988;Vali et al, 1991).…”