Imaging of Freeze-Fractured Cells with in Situ Fluorescence and Time-of-Flight Secondary Ion Mass Spectrometry

Abstract

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

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

“…20 The microscope includes two illuminator columns equipped with a 50-W halogen and a 100-W Hg light source. A Spot RT CCD camera (Diagnostic Instruments, Sterling Heights, MI) was used in monochrome mode to capture fluorescence images.…”

“…1 Numerous analytical techniques, including electrochemical detection, 2-6 separation-based methods, 7-9 fluorescence microscopy, 10-13 and mass spectrometry [14][15][16][17][18][19][20][21][22][23][24] have been used to surmount the inherent difficulties of measurements down to the μm-, pL-, and zmole-scale to study the intricate chemistry that exists within single cells.…”

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), liposomes (16,17), paramecia (18), and rat pheochromocytoma (PC12) cells (19,20), but has not yet been demonstrated for subcellular membranes. 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.…”

Chapter 13 Imaging of Cells and Tissues with Mass Spectrometry

“…The ability to control the temperature of the sample surface itself can also be of significant benefit. Cooled sample stages enable the use of frozen, hydrated samples and in situ freeze-fractured samples, both of which have shown great value in imaging experiments (Roddy et al 2002a). The frozen water acts as a matrix to enhance secondary ion yields while freeze-fracturing protocols facilitate imaging of internal structures that may not otherwise be available for study, particularly in single cell analyses.…”

“…The rapid cooling rate quenches the movement of even atomic ions such as sodium and potassium within a sample, which is indicative of the high level of preservation necessary for imaging at sub-micron spatial scales. Frozen samples may be transferred into the SIMS instrument using a cold transfer stage in a hydrated state (Roddy et al 2002a) or freeze dried to preserve the sample, keeping in mind that the drying process may allow some analyte redistribution to occur. The alternative cryogenic treatment, freeze-fracture, was originally developed for electron microscopy, and is used to access subcellular features within a sample (Roddy et al 2002b).…”