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  Research articleHigh-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometryClaude Lechene1 1National Resource for Imaging Mass Spectrometry, Harvard Medical School and Department of Medicine, Brigham and Women's Hospital, Cambridge, MA 02139, USA 2Cameca, 29 Quai des Gresillons, 92622 Gennevilliers Cedex, France 3NSee Inc., 106 Greenhaven Lane, Cary, NC 27511, USA 4Torrey Pines Institute for Molecular Studies, San Diego, CA 92121, USA 5Ocean Genome Legacy Foundation, Ipswich, MA 01938, USA 6Harvard Medical School and Renal Division, Brigham and Women's Hospital, Boston, MA 02115, USA 7Harvard Medical School, Boston, MA 02115, USA 8Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA 9Harvard Medical School and Department of Surgery, Massachusetts General Hospital, Boston, MA 02114, USA 10Universite Paris-Sud, Centre de Spectrométrie Nucléaire et de Spectrométrie de Masse, 91406 Orsay, France
Journal of Biology 2006, 5:20doi:10.1186/jbiol42 The electronic version of this article is the complete one and can be found online at: http://jbiol.com/content/5/6/20
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2006 Lechene et al.; licensee BioMed Central Ltd. AbstractBackgroundSecondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. ResultsThe new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. ConclusionMIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments. BackgroundThe fundamental discovery that proteins in biological tissues are in a dynamic state was made in the late 1930s using a custom-built mass spectrometer to measure the incorporation into proteins of the stable nitrogen isotope 15N [1], which was provided in the mouse diet as 15N-leucine and used as a marker of amino acids. These seminal studies could not be pursued at the subcellular level because there was no methodology to simultaneously image and quantitate a stable isotope and because there is no meaningful radioactive isotope of nitrogen. Imaging of stable-isotope distribution has been possible, however, since the development of mass filtered emission ion microscopy using secondary ions by Castaing and Slodzian [2], which is part of the technique later named secondary-ion mass spectrometry (SIMS). With this technique, a beam of ions (the primary-ion beam) is used as a probe to sputter the surface atomic layers of the sample into atoms or atomic clusters, a small fraction of which are ionized (Figure 1) [3]. These secondary ions, which are characteristic of the composition of the region analyzed, can be manipulated with ion optics just as visible light can be with glass lenses and prisms. In a SIMS instrument, the secondary ions are separated according to mass and then used to measure a secondary-ion current or to create a quantitative atomic mass image of the analyzed surface. SIMS has become a major tool in semiconductor and surface-science studies [4], geochemistry [5,6], the characterization of organic material [7], and cosmochemistry [8,9].
Although there has been pioneering work using SIMS in biology [10-14], SIMS technology, until now, has presented irreconcilable tradeoffs [15] that have severely limited its use as a major discovery tool in biomedical research. To make secondary-ion methodology practicable for locating and measuring isotope tags in subcellular volumes, four major issues need to be addressed. First, to produce quantitative ultrastructural images, the technique must have sufficiently high spatial resolution, and quantitation and imaging must be associated. Second, because the quantitation of label involves measuring the excess of an isotope tag above its natural occurrence, and this excess is calculated by the ratio of two isotopes, the data from the two isotopes should be recorded simultaneously, in parallel, and from exactly the same region of the sample (that is, in register). This is to ensure that changes in instrument or sample conditions do not lead to errors in the calculated isotope ratios. Third, because nitrogen has an electron affinity of zero, N- ions do not form; nitrogen must therefore be detected as cyanide ions (CN-). Consequently, in order to use the stable isotope 15N as a label, the mass resolution of the instrument needs to be high enough to separate the ions 12C15N- and 13C14N-, which have the same mass number of 27, but do not have exactly the same atomic mass weight: differing by 0.00632 mass unit, less than 1 part in 4,000. Finally, the high mass resolution should not be at the expense of the secondary-ion current; the transmission of secondary ions from sample to detector needs to be high enough to allow data collection from sub-cubic-micrometer volumes and in a reasonable amount of time. These four requirements necessitate a previously unattainable combination of instrumental capabilities, with the ability to collect large numbers of secondary ions at high mass resolution, parallel detection of several secondary ions, high lateral resolution, and high precision of measurements. A new generation of secondary-ion mass spectrometer has now been developed that can measure several ion masses in parallel, has a high mass resolution (mass/change in mass ratio of approximately 10,000) at high secondary-ion relative transmission (70–80%), and has a high lateral resolution, down to 33 nm [16,17]. In this paper we present some biological applications of this new technology. These extraordinary capabilities allow us, for example, to image and measure in parallel the intracellular distribution of molecules labeled with the stable isotopes 15N or 13C because we can separate the isobaric species (that is, the species with the same atomic masses): 12C15N- from 13C14N- or 13C- from 12C1H-. Because the images are produced in parallel from the same sputtered volume they are in exact register with each other and these characteristics are necessary for obtaining quantitative atomic mass images. A quantitative mass image contains at each pixel a number of counts, which are a measure of the selected atomic mass and are directly proportional to the selected atomic mass abundance in the sample, at a location corresponding to the pixel address. Counts from several atomic masses, originating from the same location in the sample, can be recorded in parallel at the same pixel address, which allows us to derive meaningful isotope ratios. Isotope ratios are at the core of the methodology. When the sample has been labeled with a given isotope, a ratio higher than its natural abundance indicates the presence of the marker isotope at a particular location as well as measuring its relative excess. In addition, the high stabilities of the primary beam, the ion optics, the mass spectrometer and the detectors contribute to very precise measurements. We have developed the use of this new generation of SIMS, together with tracer methods and quantitative image-analysis software, for locating and measuring molecules labeled with stable isotopes in subcellular compartments, a development that we call multi-isotope imaging mass spectrometry (MIMS). In this paper we present a range of examples showing how MIMS can be used to provide atomic mass images of biological specimens, and how in combination with stable isotope labeling it provides qualitative and quantitative information that is not possible to obtain with other methods. ResultsImaging of unlabeled cells and tissue sectionsOne qualitative application of MIMS is high-resolution imaging. Detailed anatomical images can be obtained from unstained, unlabeled samples using the 12C14N- secondary ion, as shown by the analysis of a section through mouse cochlea (Figure 2a–c). A fixed, unstained section mounted on silicon was first examined using reflection differential interference contrast (RDIC) microscopy (Figure 2a) to select regions of interest that can be retrieved after the sample is hidden inside the SIMS instrument. The mass image of the 12C14N- ions sputtered from a 80-μm field corresponding to the boxed area in Figure 2a is shown in Figure 2b, and the mass image of the 12C14N- ions sputtered from a 20-μm subfield of Figure 2b is shown in Figure 2c. The contrast of these mass images provides a very detailed view of the cochlear structures. Only a few atomic layers of the surface of the sample are sputtered using the standard analytical conditions (see Discussion and [Additional data file 1]). Thus, although the method is nominally destructive, we can analyze the same field repetitively, up to a total of tens of hours or hundreds of scans, without observing gross morphological alterations (data not shown). A large area can be imaged relatively quickly in order to select regions of interest for quantitative analysis, as illustrated by the reconstruction of a mouse cochlea shown in Figure 2d. The mass image of 12C14N- ions is made up of ten tiles, acquired over a total of 20 minutes, with each tile acquired over an area of 100 × 100 μm in 2 minutes. All the cochlear structures one would expect to see [18] are visible, and are easy to identify by comparing them with conventional histological sections.
The high spatial resolution of MIMS is illustrated by its ability to image individual stereocilia, the mechanosensory organelles of the inner hair cells of the cochlea (Figure 2e; the field analyzed was 6 × 6 μm). The various intracellular structures are easy to identify by comparing the image with electron micrographs of the same structure. We estimate that a lateral resolution better than 33 nm can be achieved using a method derived from the 'knife-edge' technique (see [Additional data file 2]). Because the atomic mass image is formed using only a few atomic layers of the sample, the depth resolution (z resolution) can be smaller than 1 nm, much better than the resolution that would be provided by an exceptionally thin electron-microscopy section (10 nm). (In this paper, we define depth resolution as the minimum amount of material that needs to be sputtered to obtain an atomic mass image.) Studies of the same sections with both MIMS and electron microscopy – a technique developed for studying cosmic dust [19] – may help provide complementary structural observations. We do not know from first principles the mechanism of contrast formation in atomic mass images from unstained samples. A striking observation of intense contrast observed with MIMS at mass 12C14N, shown in Figure 2g–i, may guide inquiries in this field. We were analyzing a mouse kidney when we observed a brightly contrasted structure, a circular 'snake', along the lining of the lumen of an artery. It is likely that this region is the 'elastica interna' as described in histology texts, a tissue layer that is usually visible only after the arterial tissue has been specially stained (Figure 2f). The MIMS images of the artery in Figure 2g–i were obtained without this special staining. The images indicate that this structure produces a high yield of 12C14N- ions in comparison with the other regions of the artery, and suggests a relationship between the image obtained and molecular composition and density. MIMS can be used to visualize whole cells as well as sections. These images have a three-dimensional appearance, showing that MIMS can provide scanning atomic or molecular ion mass images of samples with relief, as does scanning electron microscopy. The lamellipodium of a well-spread endothelial cell imaged by MIMS at mass 12C14N is shown in Figure 2j; it appears as a light, sheet-like structure with darker lines radiating from the cytoplasm to the external border of the lamellipodium. In contrast, if actin polymerization is blocked by cytochalasin D treatment, the 12C14N mass image of an endothelial cell shows no lamellipodia but only thin, spike-like projections around the cell circumference, which are most probably retraction fibers (Figure 2k). In conclusion, MIMS atomic mass images of CN- ions in biological samples are highly contrasted, even though they are obtained without any staining. The various structures are easy to identify down to a lateral resolution of approximately 33 nm, and the depth resolution can be as small as a few atomic layers. The largest single field that can be imaged is approximately 140 × 140 μm, but larger fields can be documented quickly by taking a series of images for 1–2 minutes each. There is no machine-specific requirement for the sample except that a vacuum must be sustained. Because MIMS is a surface-analysis method, one can use many kinds of samples: for example, tissue or cell sections embedded in a medium such as epon, or cells cultured directly on a support that can be brought into the analysis chamber and prepared with any usual histological technique. The thickness of the sample is not critical, provided that the electrical charges deposited can be dissipated. The surface analyzed does not have to be flat, and one can obtain SIMS images of three-dimensional samples. Quantitative labeling with stable isotopesHaving established that MIMS can be used to obtain atomic mass images of unstained biological objects, this led us to develop the unique feature of MIMS: the quantitative analysis of isotopes within subcellular compartments. We will now discuss how the technique can be used to measure the incorporation of isotopic tracers within compartments of sub-cubic-micrometer volume. To do this, the sample is labeled with stable isotopes such as 15N or 13C, which are present at much lower levels naturally than their counterpart 14N and 12C isotopes, and each isotope is then measured to determine whether it is present in an amount exceeding its natural abundance. Stable isotope labeling can be used, for example, to pursue the classic studies of Schoenheimer at the subcellular level [1]. The isotope abundance is measured by recording the secondary-ion currents (counts/time) obtained from a pair of isotopes, for example, 13C and 12C, calculating the ratio and then comparing it with their natural abundance ratio. In a control sample, which has not received an excess of the tracer isotope, the counts of each isotope are related to each other by their natural abundance. In other words, there will be a count of 13C or of 15N such that 13C/12C = 1.12%, or 15N/14N (measured as 12C15N/12C14N) = 0.367%, calculated from the values of their respective natural abundance. This means that when measured in parallel, all the analytical conditions being the same, the 15N (12C15N) count rate will be 272 times lower than the 14N (12C14N) count rate. Quantitative labeling with 15NThe goal of our first experiments was to ensure that we could measure 15N/14N ratios equivalent to their natural abundance from tiny volumes of untreated control sample. Our first analyses were carried out using a stationary cesium (Cs+) primary ion beam. We counted in parallel the secondary ions 12C14N- and 12C15N- emitted from various areas smaller than 1 square micrometer in control samples of mouse tissues. We measured 12C15N/12C14N isotope ratios in control mouse tissue of 0.366% (standard error (SE) = 0.002, n = 12) in the cochlea, 0.368% (SE = 0.001, n = 14) in the kidney, and 0.368% (SE = 0.001, n = 6) in the intestine. These values are not statistically significantly different from the natural terrestrial value of the 15N/14N isotope ratio, 0.3673% [20]. This proved the feasibility of using this method on biological samples. We then showed that we could measure the incorporation of a stable isotope label in an ultra-minute volume of biological material, as done for bulk tissue 60 years ago [1]. We fed mice a diet slightly enriched with 15N-L-leucine for a sufficient length of time (14 days) to result in total protein renewal in kidney and intestine. The 15N/14N isotope ratios determined using a stationary primary ion beam at various areas over the samples were equivalent to the 15N/14N ratio in the diet determined independently by combustion mass spectrometry analysis (intestine 4.45‰, SE = 0.05, n = 7; kidney 4.41‰, SE = 0.03, n = 12; diet 4.45‰, SE = 0.02, n = 7). Using this method, only one location can be analyzed at a time and its precise position is difficult to ascertain in the absence of an image. With our instrument, we have developed a much more powerful but more complex method of isotope ratio imaging, where the isotope ratios are calculated from quantitative mass images obtained simultaneously from a set of isotopes. A quantitative mass image, as we call it, is the representation of an analyzed field in which each pixel is the address of a register at which the secondary-ion current of an isotope of interest has been recorded during analysis. Up to four secondary-ion currents, representative of four isotopes, can be recorded simultaneously at each pixel address with our instrument, for example 12C-, 13C-, 12C14N- and 12C15N-. A quantitative image of 256 × 256 pixels thus represents a set of (256 × 256 × 4) or 262,144 numbers. We call a group of pixel addresses a 'region of interest', and the first step in quantitation is to extract the values of counts/time/isotope from groups of pixels or from individual pixels. This allows us to measure many more regions from a single analytical field than we could do using a stationary beam, and also to associate quantitation and localization among cells and subcellular domains. All the mass imaging in the rest of this paper will refer to quantitative mass imaging. We illustrate quantitative mass imaging of 15N with a study of 15N-leucine incorporation in the mouse cochlea, a highly organized tissue with several different cell types, and in a subcellular structure of this tissue, the stereocilium, the mechanosensing organelle of hair cells. The secondary-ion mass images of a field of cochlear tissue from a mouse that has been on a 15N-L-leucine diet for 9 days are shown in Figure 3a–f. Additional data file 3 describes how the quantitative data are extracted from these images. Mass images of 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. The mass image of the 12C14N- ion (Figure 3a) shows a strikingly detailed histology. 12C14N- ions arise from nitrogen-containing molecules, the most abundant by far being proteins, which make up 18% of the total weight in most cell types, whereas RNA and DNA make up 1.1% and 0.25%, respectively [21]. The mass image of the 12C15N- ions (Figure 3b) is similar in form to the 12C14N- image (Figure 3a) but has much lower counts; the total number of counts of 12C15N- ions and of 12C14N- ions are 2.02 × 105 and 4.52 × 107, respectively (note that the subjective brightness of the images is not directly related to the count rate; see Additional data file 4). The pixel count of the 12C15N- image is a measure of both natural 15N and the supplementary 15N arising from the metabolism of 15N-L-leucine in the cochlea. This supplementary 15N may vary from a minimum of zero to a maximum value equivalent to the 15N added to the diet. The image of the internal control 12C- (Figure 3d) has a relatively poor contrast compared with the 12C14N- image (Figure 3a) because a larger fraction of the 12C- ions arise from the embedding medium, which has a high and uniform carbon content. The image of the 13C- ions (Figure 3e) is similar to the 12C- image, but with a much lower count rate; the total number of counts of 13C- and of 12C- are 2.56 × 105 and 2.33 × 107, respectively. The pixel counts of the sample resulting in the 13C- ion image contain a mean of 1.10% of the 12C counts, corresponding to the natural ratio of 13C/12C.
The ratio images 12C15N-/12C14N- (Figure 3c) and 13C-/12C- (Figure 3f) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. The contrast observed in the 12C15N-/12C14N- image is due to the excess of 15N in the area of the cochlea that has incorporated 15N derived from the 15 N-L-leucine. The internal control 13C-/12C- ratio image has no contrast because, in the absence of added 13C, the value of the ratio is equivalent to the natural ratio in any part of the analyzed field. Using the quantitative images and the derived ratio images, and guided by the hue saturation intensity (HSI) transformation (see Materials and methods), we can now calculate a value for 15N incorporation into the main structures shown in Figure 3a–f. This can be expressed as percentage renewal by comparing the excess 15N in the tissue with the excess 15N in the diet (see Materials and methods). These values represent overall protein renewal among the different cochlear structures, as demonstrated for whole tissue in the classic work of Schoenheimer [1]. After 9 days of a 15N-L-leucine diet, the incorporation of 15N is markedly different among specific cell types. The outer hair cells have a 15N renewal of 52.5% ± 1.8 SD, not significantly different from that of the Deiter cells (47.2% ± 4.8 SD), or of the proximal part of the outer pillar cell above the basilar membrane (46.0% ± 5.7 SD), and of one population of tympanic border cells (48.3% ± 1.5 SD). The basilar membrane has a small 15N renewal (overall 21.1% ± 6.0 SD), not statistically different from part of the outer pillar cells at the level of the Deiter cells (18.4% ± 3.7 SD). Overall, the reticular lamina has a 15N renewal of 30.8% ± 8.9 SD, significantly higher than the basilar membrane and the distal outer pillar cells, and significantly lower than that of the outer hair cells. The lone outer hair-cell nucleus observed has a 15N renewal of 35.5%. Finally, we measured a second population of tympanic border cells with a 15N renewal significantly greater than in any other area (72.8% ± 2.5 SD). The internal control provided by the epon embedding medium had a 12C15N/12C14N isotope ratio of 0.365% ± 0.089 SD, equivalent to the natural abundance ratio and corresponding to a 15N renewal of 0%. The unique power of MIMS is demonstrated by the quantitative imaging of subcellular structures at high resolution, revealing sub-cubic-micrometer-sized zones with high 15N renewal, and thus probably high protein renewal. In addition, the experiment showed that the same sample can be analyzed repetitively at a variety of spatial resolutions. We analyzed one of the bundles of stereocilia (Sb1, indicated by a white arrow in Figure 3a) at high resolution; we used a field of 3 × 3 μm, a beam size of about 35 nm, and 256 × 256 pixels (Figure 3g–l). Mass images of the 12C-, 13C-, 12 C14N- and 12C15N- ions were acquired in parallel. The 12C14N- image (Figure 3g) shows one bundle of stereocilia and a fraction of the cuticular plate of one hair cell, barely visible in the lower-resolution image in Figure 3a. As in the cochlear analysis, but at a subcellular level, the 12C15N- image (Figure 3h) is similar in form to the 12C14N- image (Figure 3g) but has much lower counts; the total number of counts of 12C15N- and of 12C14N- are 8.36 × 104 and 1.61 × 107, respectively. The 12C- image (Figure 3j) has relatively poor contrast compared with the 12C14N- image (Figure 3g), as most of the 12C- ions arise from the embedding medium. The 13C- image (Figure 3k) is similar to the 12C- image, but with a much lower count; the total number of counts for 13C- and 12C- are 4.80 × 105 and 4.36 × 107, respectively. The pixel counts from the 13C- image include the fraction of 13C related to the 12C content by the natural ratio of 13C/12C. The ratio images 12C15N-/12C14N- (Figure 3i) and 13C-/12C- (Figure 3l) result from the pixel-by-pixel division of the 12C15N- image by the 12C14N- image and of the 13C- image by the 12C- image, respectively. The contrast observed in the 12C15N-/12C14N- image is due to the excess of 15N in the stereocilia, cuticular plate, and hair-cell areas that have incorporated 15N derived from the 15N-L-leucine. The internal control 13C-/12C- ratio image (Figure 3l) has no contrast, as in Figure 3f. An HSI transformation of the 12C15N-/12C14N- ratio image of the stereocilia bundle in Figure 3i is shown in Figure 3m. The colors indicate the fractional excess 15N derived from the measured 12C15N-/12C14N- isotope ratios. The HSI image reveals small areas of high excess 15N located towards the tips of stereocilia or between stereocilia (magenta); within the stereocilia, close to these areas, there is minimal or no excess 15N, as indicated by the predominantly blue-green to blue color. Guided by the HSI image, we have calculated the values of the 12C15N-/12C14N- ratios and of the percentage 15N renewal for the areas indicated α to ζ and at 100 to 200 nm away from them within the stereocilia core over an approximately equivalent area (Figure 3m and Table 1). We measured high 15N renewal in areas α to ζ (79.4% ± 12.7 SE, n = 5), whereas at 200 nm away the 15N renewal in stereocilia was very low (4.6% ± 1.27 SE, n = 5). Finally, MIMS allowed us to estimate from the relative counting of mass 12C14N in areas α to ζ and in stereocilia that the above values may have been produced by objects about 5 nm wide (see also Figure 5k below in the section entitled 'Quantitative labeling of prokaryotic with gaseous 15N). The overall mean values of 15N renewal in structures between stereocilia, found with HSI, and in adjacent stereocilium cores are shown in Figure 3n. After 9 days on the 15N-L-leucine diet, the mean 15N incorporation into the inter-stereocilia structures was 78.6% ± 10.1 SE (n = 7). In the adjacent stereocilium core (200 nm away), the 15N incorporation was 7.1% ± 2.1 SE (n = 7). After 22 days on the 15N-L-leucine diet, the incorporation of 15N into the inter-stereocilia structures was 100% of its content in the diet, and in the adjacent stereocilium cores, 15N incorporation was 20.9% ± 3.8 SE (n = 4). In the areas in which 15N values were very different, the internal control 13C/12C ratios (Figure 3o) were very similar between inter-stereocilia structures (1.09% ± 0.04 SE, n = 7) and adjacent stereocilium cores (1.12% ± 0.03 SE, n = 7), and are statistically equivalent to the natural terrestrial ratio of 1.12% [20].
Table 1. Calculated percent nitrogen renewal from stereocilia regions analyzed in Figure 3m We can thus measure with high precision in a single analyzed field a variety of values of 15N incorporation among different cell types, as calculated from the quantitative mass images in Figure 3a–c, or among subcellular structure over an area of 9 μm2 square, as calculated from the quantitative mass images in Figure 3g–i. Quantitative labeling with 13CDespite the importance of free fatty acids (FFAs) for life, studies of their transport are difficult to extend to the cellular scale because no suitable methodology is available. Autoradiography cannot provide quantitative information on accumulation of FFAs in intracellular fat droplets, and fluorescently labeled FFAs may not accurately reflect the transport and metabolism of native FFAs [22,23]. As a result, the mechanism that transports FFAs across a cell membrane remains uncertain (for recent reviews see [24-27]). Using quantitative mass imaging with MIMS we have directly studied the accumulation of 13C in cultured adipocytes incubated with 13C-labeled oleic acid (13C-OA; see [28] for further details). We measured a high level of 13C accumulation in intracellular lipid droplets. Quantitative MIMS images were obtained in parallel for 12C-, 13C-, 12C14N-, and the isobars 13C14N- and 12C15N-. The relative excess of 13C was measured at three different locations: outside the cell, inside the cell but outside the lipid droplets, and inside the lipid droplets. The quantitative mass images of an adipocyte exposed to 13C-OA for 20 minutes are shown in Figure 4a–i. Images of the 12C-, 13C-, 12C14N- and 12C15N- ions or of the 12C-, 13C-, 12C14N- and 13C14N- ions were acquired in parallel. Images of the 12C- and 12C14N- ions (Figure 4a,d) show the cell histology. The mass image of the 13C- ion (Figure 4b) is similar to the 12C- ion image (Figure 4a) in form, but has a lower count rate. The pixel counts of the 13C- image include both the natural 13C and the supplementary 13C from the 13C-OA transported into the cell. This supplementary 13C is at a maximum at the intracellular lipid droplets, where FFAs accumulate. The mass image of the internal control 12C15N- ions (Figure 4e) is similar to the 12C14N- mass image, yet with a much lower count rate. Each pixel of the sample resulting in the 12C15N- image contains the fraction of 15N related to the 14N content by the natural ratio of 15N/14N. The enhanced contrast observed in the 13C-/12C- image (Figure 4c) is due to the excess 13C incorporated into the lipid droplets from the transported 13C-OA. The internal control 12C15N-/12C14N- ratio image (Figure 4f) has no contrast because in the absence of added 15N, the value of the ratio of 15N/14N is equivalent to the natural ratio across the analyzed field.
The HSI image of the 13C-/12C- ratio is shown in Figure 4g, and the same cell photographed by RDIC microscopy on the silicon chip before analysis with MIMS is shown in Figure 4h. The 13C-/12C- ratio, indirectly measured as the cyanide ion, 13C14N-/12C14N-, is shown in Figure 4i; this also shows accumulation of 13C- in the droplets. In contrast to the high 13C-/12C- ratios found in cells that were incubated with 13C-OA, cells washed with buffer solution after 13C-OA incubation had low 13C-/12C- ratios (images not shown). In cells not treated with 13C-OA, the value of the 13C-/12C- ratio measured under the same conditions was 1.15 ± 0.10%, not significantly different from the terrestrial 13C/12C value of 1.12%. An indication of the accuracy of these values was obtained from measurements of the 12C15N-/12C14N- ratio, whose value was 0.36 ± 0.01% in both washed and unwashed cells, in excellent agreement with the natural abundance of 0.37%. The cumulative values obtained from quantitative MIMS atomic mass images and extracted from the isotope ratio images are shown in Figure 4k. We can remove material quickly from the sample surface in order to study a variety of depths within the cell. We refer to this as 'shaving' the sample. It is accomplished in conditions that give a high primary-ion beam current (such as by removing the objective diaphragm; see Figure 13 in the Discussion section). The results of such shaving are shown in Figure 4j. The adipocyte analyzed in Figure 4a–i was shaved using, for a few minutes, a primary beam current approximately 1,000-fold more intense than for the previous analysis. This uncovered a lipid droplet deeper in the cell with a very high 13C-/12C- ratio, as shown in the HSI image (Figure 4j). Finally, MIMS allows us to acquire hundreds of atomic mass image planes successively from the same cell, opening the door to full three-dimensional volume rendering. We have begun using this capability to study the distribution of 13C among the lipid droplets located within a single adipocyte after incubation with 13C-OA [29]. In conclusion, MIMS can be used to investigate lipid metabolism with high spatial and quantitative resolution [28]. Unlike other techniques, MIMS allows us to trace and to measure the movement of native FFAs at specific subcellular locations. Quantitative labeling of prokaryotic cells with gaseous 15NThe ability to image and measure stable isotopes makes it easy and safe to apply MIMS to samples labeled with a gaseous precursor. Here we describe the application of MIMS to the study of nitrogen fixation in bacteria (Figure 5a–k). Teredinibacter turnerae is a diazotrophic (nitrogen-fixing) marine bacterium that can be isolated from the tissues of wood-boring marine bivalves (family Teredinidae) and grown in pure culture [30,31]. Enterococcus faecalis is a bacterium that does not fix nitrogen. Both were cultured for 120 hours in a 15N atmosphere. Mass images of the 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. T. turnerae is barely visible at mass 12C14N- (Figure 5a) but is seen as intensely labeled at mass 12C15N- (Figure 5b) because it has used gaseous 15N to build its molecular constituents. By contrast, E. faecalis is visible at mass 12C14N- (Figure 5a) but not at mass 12C15N- (Figure 5b) because it does not use gaseous nitrogen and therefore has not incorporated 15N above the natural ratio. The HSI image of the 12C15N-/12C14N- ratio (Figure 5c) shows E. faecalis with an isotope ratio equivalent to the natural isotope ratio (blue) and T. turnerae, which has incorporated an enormous quantity of 15N, with an isotope ratio at least 100 times higher (magenta). MIMS can also be used to study the distribution of isotope tag incorporation within a bacterial population. The heterogeneity of nitrogen fixation among a population of T. turnerae is shown in Figure 5d, where the same field cultured for 32 hours in a 15N atmosphere is shown as a series of HSI 12C15N-/12C14N- ratio images. The different HSI panels reveal the level of 15N incorporation in bacteria using a compressed color scale as described in the legend to Figure 5. This analysis reveals the location and the distribution of ratio values, in other words of nitrogen fixation, among the bacteria within the analyzed field. Large differences in the amounts of 15N incorporation by T. turnerae cultured for 96 hours in a 15N atmosphere are demonstrated by the HSI images of the 12C15N-/12C14N- ratio (Figure 5e,f). Differences are visible among a few bacteria in contact with each other (Figure 5e) and even within a single bacterium (Figure 5f). MIMS can detect and measure the function of intracellular bacteria within eukaryotic cells. This is shown by the quantitative imaging of the incorporation of 15N in T. turnerae living in the gill bacteriocytes of a shipworm (Lyrodus pedicellatus) raised under a 15N atmosphere, as shown in the HSI image of the 12C15N-/12C14N- ratio (Figure 5g). The bacteria in the bacteriocytes that have incorporated 15N are shown in colors between yellow and magenta (the shipworm tissue is blue). An internal control is the isotope ratio 13C-/12C- of the same field (Figure 5h), which shows a lack of contrast. The uniformity of the carbon ratio image eliminates the possibility of artifacts in the nitrogen ratio image as a result of morphologically or instrumentally induced isotope fractionation. These quantitative images show that the MIMS method will be a powerful tool in the investigation of nitrogen fixation. It can also be used to study bacteria in natural environments and to explore the activity of diazotrophic symbionts in the tissues of plants and animals. It is worth noting that the size of an object can be estimated directly from the pixel signal intensity. This is illustrated with the flagellum visible on one T. turnerae cell (Figure 5i). For example, at mass 12C15N (Figure 5j), we have a mean of 1,473 counts per pixel on the bacterium (Figure 5k, inset, red box). A line profile of the counts per pixel across the flagellum of T. turnerae at mass 12C15N is shown in Figure 5k. The pixel crossed by the flagellum registered 66 counts. All conditions being approximately the same, the number of counts is proportional to the surface area of the material sampled in one pixel. In this particular image of 60 × 60 μm, 256 × 256 pixels, one pixel is equivalent to 234 nm covering an area of 54,756 nm2. If the length of the flagellum crosses a pixel, 66 counts would represent a width of (54,756 nm2/1,473 counts) × (66 counts/234 nm) = 10.5 nm; using the count values for the same pixels at mass 12C14N, the estimate is 10.6 nm, which is approximately the diameter of a T. turnerae flagellum. Use of double labeling with bromodeoxyuridine and 15N-leucine to measure protein renewalBecause MIMS analysis sputters only a few atomic layers, a sample can be reanalyzed many times. This is illustrated by double-labeling studies of protein renewal and DNA replication in the mouse kidney after ischemia. We have previously shown [32] in the mouse that 30 minutes of bilateral renal ischemia, resulting in significant increases of blood urea nitrogen and creatinine, leads to protection of the mouse kidney against a subsequent ischemic insult 8 or 15 days later, even when the second ischemic period is extended to 35 minutes. Graded levels of time of initial ischemia resulted in graded levels of protection 8 days later. Bromodeoxyuridine (BrdU) and 15N-leucine were administered to mice subsequent to the first ischemia, in order to characterize the different response in cell proliferation in preconditioned and non-preconditioned kidneys exposed to ischemia on day 8 after the initial surgery. Quantitative MIMS images were recorded from thin sections of epon-embedded kidneys. The images were acquired in parallel at mass 12C14N to show a morphological overview, at mass 31P to view the cell nuclei and at mass 81Br to identify the nuclei undergoing DNA replication, as shown by BrdU incorporation. Protein renewal was calculated from parallel imaging at mass 12C14N and mass 12C15N. A first MIMS analysis of a 100 × 100 μm field for 2 minutes, as shown in Figure 6a,b, reveals that one nucleus (in the 81Br- image in Figure 6b) has replicated its DNA, whereas the others have not. A second MIMS analysis at higher resolution of cells around this replicating nucleus is shown in Figure 6c–e. The replicating nucleus is seen in the 81Br- mass image (Figure 6e), and the 31P- image (Figure 6d) shows the presence of two other nuclei that did not replicate. A third MIMS analysis was performed on the same field at masses 12C14N (Figure 6f) and 12C15N (Figure 6g) to quantitate the protein renewal and compare renewal between replicating and non-replicating cells. We found that incorporation of 15N into the replicating nuclei was twice as high as that in either the cytoplasm or in non-replicating cells (nuclei or cytoplasm; Figure 6h). The 12C14N images acquired successively (Figure 6c,f) show that there are no visible changes; this validates the comparison of data obtained in the second and third analysis. MIMS can thus be used with multiple tags that can be studied with a succession of parallel analyses of the same field at a variety of isotope combinations.
Qualitative labeling with stable isotopesMIMS methodology enables us to study the spatial aspects of metabolic pathways and the spatial relationship between replication and transcription. As an example, we will show MIMS atomic mass imaging of the co-localization of RNA and DNA. Rat embryo fibroblasts were pulsed with 15N-uridine and BrdU, markers of newly synthesized RNA and DNA, respectively. The simultaneously recorded distributions of 12C15N- and 81Br- are shown in Figure 7a,b. As expected, the bromine signal (DNA) is restricted to the cell nuclei; there is strong 81Br labeling along the nuclear envelope and around the nucleoli (Figure 7b). In contrast, the 12C15N- signal (RNA) is strong within the nucleoli and along the nuclear envelope (Figure 7a).
An overlay of the 12C15N- signal in red with the 81Br- signal in green shows the co-localization of newly synthesized RNA and DNA in yellow (Figure 7c). This co-localization, visualized directly from the isotope images, avoids the complications potentially introduced by immunochemical methods [33]. Localization of newly synthesized RNA requires distinguishing a local excess of 15N over its natural occurrence. We carried out another MIMS analysis of the same cells to record the distributions of 12C14N- and 12C15N- in parallel (in our prototype instrument, we cannot simultaneously detect the isobars 12C14N- and 12C15N- together with 81Br- because of the steric hindrance of the electron multipliers). Overlaying the 12C14N- signal in red with the 12C15N- signal in green shows up the local excesses of 15N above its natural occurrence in yellow (Figure 7d). The yellow identifies the localization of 15N-uridine-labeled newly synthesized RNA within the nucleoli, along the nuclear envelope, and in the cytoplasm of the top cell. The importance of parallel detection and isotope ratio imaging is illustrated by the following fortuitous observation. We used MIMS to study the distribution of RNA in the nucleolus by studying fibroblasts cultured in the presence of 15 N-uridine. Quantitative mass images of 12C-, 12C14N- and 12C15N- secondary ions were acquired in parallel. In the fibroblast shown in Figure 8, the nuclear membrane is clearly visible at mass 12C14N- (Figure 8a), and two nucleoli are seen highly contrasted at masses 12C14N- and 12C15N- (Figure 8a,b). Higher resolution parallel mass images of the nucleolus seen on the right in Figure 8a are shown in Figure 8c–e. In these cells embedded in epon – a polymer lacking nitrogen – the 12C- image (Figure 8c) shows little contrast except for a dark spot (diameter around 122 nm) in the middle (Figure 8c, red arrow; the low brightness indicates low counts). This spot was caused by accidental exposure of the sample to an intense stationary primary-ion beam. The spot is also seen in the 12C14N- and the 12C15N- images (Figure 8d,e). The 12C15N- image, however, contains four additional dark regions (Figure 8e, white arrows), ranging from 200 to 280 nm in diameter. Nevertheless, isotope ratio and HSI derivation of 12C15N- and 12C14N- images clearly distinguish the accidental dark spot, which has a high level of 15N incorporation, from the other four sub-nucleolar regions, which have low 15N incorporation; they can therefore be taken to be related to nucleolar organization (Figure 8f,g). We also used MIMS to evaluate the dose response of uridine incorporation in nucleoli of rat embryo fibroblasts cultured in the presence of 0.0, 0.01, 0.1, and 1.0 mM 15N-uridine (Figure 8h), demonstrating that MIMS may be used to establish a dose-response curve at the level of intracellular organelles.
Use of MIMS without isotope labeling to study gross differences in subcellular compositionQuantitative mass images of the chemical elements within a cell can provide information on the existence and location of subcellular domains with gross differences in composition. Thus, even without exposing the cells or tissues to isotopically labeled molecules, we may obtain a measure of the gross cellular composition at the level of microdomains that cover areas of sub-micrometer size. This is illustrated by the overlay image of an endothelial cell analyzed in parallel for 12C-, 12C14N- and 31P- (Figure 9). Striking differences in gross composition within a cell are revealed. The area over the nucleus and the thicker part of the cytoplasm is intensely red, an indication of comparatively high nitrogen content; the wide area at the periphery (the lamellipodium) is relatively rich in phosphorus and poor in nitrogen; and at the outmost edge of the cell, there is relatively more carbon than in the wide part of the lamellipodium. Short, thin protrusions with a relatively high nitrogen signal can also be seen at the very edge of the cells; these are probably filopodia. One may assume the following: high 12C14N indicates protein (or glycoproteins); high 12C14N associated with phosphorus indicates nucleotides; 12C with less 12C14N indicates lipids or sugars; and 12C associated with 31P indicates phospholipids.
We undertook a more detailed analysis of the region at the edge of the lamellipodia of endothelial cells (see Figure 9), in which we saw domains two pixels wide (the pixel size here is 234 nm) containing five times more nitrogen and a tenth the level of oxygen than the neighboring pixels. From the original MIMS images acquired in parallel at masses 12C14N-, 12C- and 16O- (Figure 10a–c), together with HSI images of the ratio 12C14N-/12C- (Figure 10d) and of the ratio 16O-/12C14N- (not shown), we observe lamellipodial domains at the edge of the cell that look like regularly spaced 'dots'. These are rich in 12C14N and poor in 16O compared with their surroundings. The values of 12C14N- counts, 12C- counts and of the 12C14N-/12C- ratio are shown in Figure 10f for a group of pixels constituting the central dot of the inset in Figure 10e, and the values of the 16O-/12C14N- and of the 12C14N-/12C- ratios are shown in Figure 10g for the same pixels; these panels illustrate the way in which quantitation and imaging are intimately associated in MIMS. The white rectangle in Figure 10f,g surrounds two neighboring pixels that have the highest nitrogen content and the lowest oxygen content (Figure 10h) compared with the surrounding lamellipodium (Figure 10i). Thus, parallel quantitative mass imaging using MIMS without isotopic supplementation can identify nanometer-sized structures that may be functionally significant.
Labeling with radioactive 14CMIMS opens the world of stable isotopes to quantitative nanoautography. It is similar in principle but much more powerful than autoradiography because it is precisely quantitative, needs much shorter exposure times, can use the very large number of stable isotopes that are naturally available, can easily use multiple labels, gives high lateral resolutions, provides exceptional depth resolution and would be harmless for clinical use. MIMS can also be used for high-resolution quantitative imaging of radioisotopes (such as 14C), and with high sensitivity, as shown in the pioneering work of Hindie et al. [10]. An example of parallel imaging at masses 14C- and 12C15N- of a whole fibroblast pulsed with serum and then with 14C-thymidine after serum deprivation is shown in Figure 11a,b, and the overlay of the 12C15N- and 14C- images in Figure 11c. The 12C15N- and 14C- images from a control sample with no added 14C are shown in Figure 11d,e. The 12C15N- mass image maps the whole fibroblast (Figure 11a). The 14C- atomic mass image, indicative of the 14C-thymidine in the DNA, is restricted to the nucleus (mean count within nucleus = 49.0, SD = 38.2, n pixels = 2,388, sum of counts = 116,955). The 14C signal is variable across the nucleus and is segregated into domains, reminiscent of the results of Wei et al. [34]. The mean background 14C count outside the fibroblast is 0.6 (SD = 1.1, n pixels = 31,148, sum of counts = 19,873). The mean 14C count in the nuclear region of the control sample is 0.009 (SD = 0.128, n pixels = 2,875, sum of counts = 25). The mean 14C count outside the nucleus of the control sample is not significantly different, with a value of 0.008 (SD = 0.153, n pixels = 50,842, sum of counts = 420). Thus after exposure to 19 nmol/ml of 14C-thymidine, counts in the nucleus of the labeled sample have increased by a factor of 6,125 relative to the control sample. Control background count of 14C is negligible. Compared with autoradiography, one can estimate that even if only 1‰ of the 14C atoms were sputtered and only 1% of the sputtered atoms were ionized, the sensitivity of MIMS is at least 103-fold greater (see Additional data file 5 for calculations).
Use of MIMS to track individual cells in large cell populationsFollowing the transplantation of allogeneic tissues and organs, some donor-derived cells are known to leave the graft and present alloantigens to the host's immune system [35-37]. The exact nature and frequency of the donor cells infiltrating the host's lymphoid tissues are still unclear, however. The movement patterns of recipient immuno-competent cells that recognize the alloantigens are also unknown. These questions have remained unanswered because of the lack of sufficiently sensitive techniques for accurately tracking and visualizing small numbers of lymphocytes in tissues. Here, we use MIMS to track donor cells after transplantation. Spleen cells from C57Bl/6 (B6, MHC haplotype H-2b) mice fed for 2 weeks with a 15N diet were injected subcutaneously in the footpad of a fully allogeneic BALB/c mouse (H-2d). Two days later, popliteal lymph nodes (located behind the knee, where they drain the foot pad of the mouse) were collected and examined by MIMS for the presence of donor cells. Mass images of the 12C-, 13C-, 12C14N- and 12C15N- ions were acquired in parallel. Because only a very few donor cells are expected to be found among the recipient cells, a large number of cells need to be examined relatively quickly with some means of highlighting the areas that could contain donor cell(s). This was done using MIMS to rapidly map a large area of lymph node as shown in Figure 12a,b. We clearly observe areas where there appears to be a small excess of 15N above its natural abundance (Figure 12b). These areas were then reanalyzed with MIMS at a higher resolution over a longer time. A field containing donor cells is shown in Figure 12c–e. In the 12C15N-/12C14N- ratio image (Figure 12d), two donor cells are clearly visible. The ratio image of 13C-/12C- of the same field taken simultaneously (Figure 12e) does not show any contrast and acts as an internal control. The frequency of transplanted cells infiltrating the recipient lymph node was estimated to be 690 cells per million.
We conclude that the MIMS technique allows the detection and frequency determination of rare cells infiltrating the recipient's lymphoid tissues after transplantation. The methodology can be extended to study solid organ transplants and the nesting of stem cells. It is important to note that the current techniques for tracking transplanted cells are fraught with difficulties [38]. Labeling DNA with stable isotopes should provide a method allowing long-term and physiological marking, the possibility of determining the generation of single cells from the amount of label within the cell, and the possibility of labeling both donor and recipient cells differently, enabling the unambiguous recognition of cell fusion or phagocytosis of donor cells by the recipient's cells [38]. DiscussionWe have shown that we can obtain quantitative atomic mass images using MIMS. Because these images are produced in parallel from the same sputtered volume they are in exact register with each other, which is necessary to derive meaningful isotope ratios. A lateral resolution that reaches 33 nm, in theory, and a high mass resolution (a mass/change in mass ratio of approximately 10,000) at high secondary-ion transmission (70–80%) allows us to image and quantitate molecules labeled with stable (or radioactive) isotopes within subcellular compartments. The high mass resolution allows us to distinguish the distribution of isobaric atomic species. For example, by separating 12C15N- from 13C14N- and 13C- from 12C1H-, we can image the intracellular distribution of molecules labeled with the stable isotopes 13C and 15N (such as 15N-thymidine and 13 C-uridine) in parallel and in the same preparation. Finally, the high stability of the primary beam, the mass spectrometer and the detectors contribute to precise quantification. These unique characteristics have allowed us to develop MIMS. Instrumental advancesInstrument modificationsIn SIMS, a primary-ion beam (usually Cs+ or O-) is used as a probe to scan across the surface of a solid sample. The impact of a primary ion on the surface of the sample triggers a cascade of atomic collisions, and atoms and clusters of atoms are ejected, most originating in the neighborhood of the impact point. In the ejection process, some of them will be spontaneously ionized; these secondary ions are characteristic of the composition of the target area (see Figure 1). After separating the secondary ions acc |
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