Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy

Abstract

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.

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.…”

Preparation of Single Cells for Imaging Mass Spectrometry

“…The freeze-fracture technique was originally reported in 1957 and has been used extensively to prepare cells for membrane analysis by electron microscopy [19,20]. Working from this concept, Chandra and coworkers developed a modified freeze-fracture method that could be used to prepare cells for imaging MS analysis [21]. Currently, the vast majority of cellular MS imaging reports utilize some variation on this method.…”

Peer Reviewed: A Subcellular Imaging by Dynamic SIMS Ion Microscopy.

“…To minimize this damage, sample freezing and freezedrying at low temperatures are desirable. For example, morphological evaluations of fractured cells and membrane pieces prepared with our "sandwich fracture" method (24), using electron and laser scanning confocal microscopic techniques, revealed well-preserved membrane particles, mitochondria, lysosomes, Golgi apparatus, and cell cytoskeleton (24)(25)(26)(27). This is not surprising, because the method quickfreezes cell monolayers of <10-µm thickness and freeze-dries the sample at temperatures of -80 °C or lower (24).…”