| 1. |
- Piehowski, Paul D., et al.
(author)
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Freeze-Etching and Vapor Matrix Deposition for ToF-SIMS Imaging of Single Cells
- 2008
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 24:15, s. 7906-7911
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Journal article (peer-reviewed)abstract
- Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has been extensively used in conjunction with electron microscopy. Here, we apply this technique to time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. We demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, we found water can be controllably redeposited onto the sample at temperatures below −100 °C in vacuum. The redeposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, we have obtained high quality spectra with both atomic bombardment as well as C60+ cluster ion bombardment.
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| 2. |
- Piehowski, Paul D., et al.
(author)
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MS/MS Methodology To Improve Subcellular Mapping of Cholesterol Using TOF-SIMS
- 2008
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In: Anal. Chem.. - : American Chemical Society (ACS). ; 80:22, s. 8662-8667
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Journal article (peer-reviewed)abstract
- Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60+/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.
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| 3. |
- Piehowski, Paul D, et al.
(author)
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Time-of-flight secondary ion mass spectrometry imaging of subcellular lipid heterogeneity: Poisson counting and spatial resolution.
- 2009
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In: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:14, s. 5593-602
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Journal article (peer-reviewed)abstract
- Mass spectrometric imaging is a powerful tool to interrogate biological complexity. One such technique, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging, has been successfully utilized for subcellular imaging of cell membrane components. In order for this technique to provide insight into biological processes, it is critical to characterize the figures of merit. Because a SIMS instrument counts individual events, the precision of the measurement is controlled by counting statistics. As the analysis area decreases, the number of molecules available for analysis diminishes. This becomes critical when imaging subcellular features; it limits the information obtainable, resulting in images with only a few counts of interest per pixel. Many features observed in low intensity images are artifacts of counting statistics, making validation of these features crucial to arriving at accurate conclusions. With TOF-SIMS imaging, the experimentally attainable spatial resolution is a function of the molecule of interest, sample matrix, concentration, primary ion, instrument transmission, and spot size of the primary ion beam. A model, based on Poisson statistics, has been developed to validate SIMS imaging data when signal is limited. This model can be used to estimate the effective spatial resolution and limits of detection prior to analysis, making it a powerful tool for tailoring future investigations. In addition, the model allows comparison of pixel-to-pixel intensity and can be used to validate the significance of observed image features. The implications and capabilities of the model are demonstrated by imaging the cell membrane of resting RBL-2H3 mast cells.
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| 4. |
- Ding, J., et al.
(author)
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Inhibition of HMGCoA Reductase Reveals An Unexpected Role for Cholesterol During PGC Migration in the Mouse.
- 2008
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In: BMC developmental biology. - 1471-213X. ; 8:1
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Journal article (peer-reviewed)abstract
- ABSTRACT: BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSIONS: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.
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| 5. |
- Kurczy, Michael, 1980, et al.
(author)
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Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging
- 2008
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In: Applied Surface Science. - : Elsevier BV. - 0169-4332. ; 255:4, s. 1158-1161
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Journal article (peer-reviewed)abstract
- We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C60+ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.
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| 6. |
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| 7. |
- Ostrowski, Sara G, et al.
(author)
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Secondary Ion MS Imaging To Relatively Quantify Cholesterol in the Membranes of Individual Cells from Differentially Treated Populations
- 2007
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In: Analytical chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 79:10, s. 3554-3560
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Journal article (peer-reviewed)abstract
- Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a well-established bioanalytical method for directly imaging the chemical distribution across single cells. Here we report a protocol for the use of SIMS imaging to comparatively quantify the relative difference in cholesterol level between the plasma membranes of two cells. It should be possible to apply this procedure to the study of other selected lipids. This development enables direct comparison of the chemical effects of different drug treatments and incubation conditions in the plasma membrane at the single-cell level. Relative, quantitative TOF-SIMS imaging has been used here to compare macrophage cells treated to contain elevated levels of cholesterol with respect to control cells. In situ fluorescence microscopy with two different membrane dyes was used to discriminate morphologically similar but differentially treated cells prior to SIMS analysis. SIMS images of fluorescently identified cells reveal that the two populations of cells have distinct outer leaflet membrane compositions with the membranes of the cholesterol-treated macrophages containing more than twice the amount of cholesterol of control macrophages. Relative quantification with SIMS to compare the chemical composition of single cells can provide valuable information about normal biological functions, causative agents of diseases, and possible therapies for diseases.
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