Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

X-ray spectroscopy and imaging of selenium in living systems

Background

Selenium is an essential element with a rich and varied chemistry in living organisms. It plays a variety of important roles ranging from being essential in enzymes that are critical for redox homeostasis to acting as a deterrent for herbivory in hyperaccumulating plants. Despite its importance there are many open questions, especially related to its chemistry in situ within living organisms.

Examination of trafficking of phagocytosed colloid particles in neutrophils using synchrotron-based X-ray fluorescence microscopy (XFM)

Synchrotron-based X-ray fluorescence microscopy (XFM) can localise chemical elements at a subcellular level. 99mTechnetium stannous (TcSn) colloid is taken up by phagocytes via a Complement Receptor 3 mediated phagocytic process. In the current study, XFM was used to examine the intracellular trafficking of TcSn colloid in neutrophils. XFM was performed on TcSn colloid, and neutrophils labelled with TcSn colloid, in whole blood. We developed a set of pixel by pixel analysis and mapping techniques incorporating cluster analysis that allowed us to differentiate neutrophils and artefactual contaminants, and we examined the changes in element distribution that accompany neutrophil phagocytosis of TcSn colloid. Sn became associated with half the neutrophils. Within cells, Sn colocalised with iron (Fe) and sulphur (S), and was negatively associated with calcium (Ca). Despite the high sensitivity of XFM, Tc was not detected. XFM can help clarify the intracellular processes that accompany neutrophil phagocytosis. The subcellular colocalisation of Sn with Fe is consistent with fusion of the colloid-containing phagosome with neutrophil granules. The association of Sn with S suggests that proteins rich in S-containing amino acids are present in the phagosome. The negative colocalisation with Ca indicates that ongoing maturation of the TcSn colloid phagosome is no longer calcium dependent one hour after phagocytosis.     

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.     

Quantitative permeability imaging of plant tissues

A method for mapping tissue permeability based on time-dependent diffusion measurements is presented. A pulsed field gradient sequence to measure the diffusion encoding time dependence of the diffusion coefficients based on the detection of stimulated spin echoes to enable long diffusion times is combined with a turbo spin echo sequence for fast NMR imaging (MRI). A fitting function is suggested to describe the time dependence of the apparent diffusion constant in porous (bio-)materials, even if the time range of the apparent diffusion coefficient is limited due to relaxation of the magnetization. The method is demonstrated by characterizing anisotropic cell dimensions and permeability on a subpixel level of different tissues of a carrot (Daucus carota) taproot in the radial and axial directions.     

Chemical sectioning fluorescence tomography: high-throughput,high-contrast,multicolor, whole-brain imaging at subcellular resolution

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Elemental Analysis in Murine Central Nervous System: Elevation of Rubidium Subsequent to Newcastle Disease Virus Encephalopathy

The Cg strain of Newcastle disease virus (NDV) produces neurologic signs and death in mice. This illness is unusual because of the lack of typical features of a viral encephalitis. Specifically, there is a paucity of infectious virus, detectable cellular inflammatory reaction, cytopathic effect, and viral antigen by immunofluorescence. We previously showed an elevation of alpha-aminoisobutyric acid in the CNS of moribund NDV-infected mice, indicating cellular membrane dysfunction. In an attempt to further our understanding of the pathogenesis of the illness, we evaluated CNS concentrations of sodium, potassium, iron, copper, zinc, magnesium, selenium, and rubidium. Elemental analysis revealed no difference between infected and control mice for all elements except for rubidium, which was significantly elevated in infected mice. Elevation in rubidium was detected in infected mice by X-ray fluorescence and atomic absorption spectrophotometry, whereas rubidium concentrations for control mice were similar by both methods. Neurologic symptoms correlated directly with rising rubidium concentrations. Our data suggest that abnormal trace element levels during viral infection may be one mechanism responsible for the clinical symptoms.     

X-ray microanalysis of chromatin-bound period IV metals inGlenodinium foliaceum: A binucleate dinoflagellate

Summary Each vegetative cell of the dinoflagellateGlenodinium foliaceum possesses two distinct types of nucleus, both of which have high levels of chromatin-bound Period IV (Periodic Table) metal elements.The typical dinoflagellate (dinocaryotic) nucleus has chromatin which differs from the atypical (supernumerary) nucleus in its high degree of condensation and in the related high levels of P, Ca, and Transition metals Fe, Ni, Cu, and Zn. The complete absence of detectable Fe and Ni in the supernumerary chromatin represents a major difference which may relate to differences in phyllogenetic origin of the two nuclei.The two types of chromatin show close similarities at the molecular level, including the possession of 40 atoms of Period IV elements per 100 atoms of P—of which approximately half are Ca atoms, and half Transition metals. In both cases, the levels of Ca and Zn show a high correlation with the level of P, suggesting a direct association of these particular metal atoms with nucleic acid phosphate groups. The close similarity in metal binding at the molecular level suggests that the association of Period IV elements with the two types of chromatin is unrelated to any differences in chromatin proteins—such as the presence or absence of histones.     

Quantitative bioluminescence imaging of transgene expression in vivo

Bioluminescent imaging (BLI) is a widely used in vivo method to determine the location and relative intensity of luciferase expression in mice. Luciferase expression is observed following an i.p. dose of d-luciferin, resulting in bioluminescence that is detected in anesthetized mice by a charge-coupled device camera. To establish whether BLI could be used as a quantitative measurement of non-viral-mediated luciferase expression, precise quantities of plasmid DNA encoding the luciferase gene were hydrodynamically dosed in mice. The results established a linear correlation between the DNA dose and the BLI response measured in liver which spanned five orders of magnitude. The level of luciferase expression was found to be a direct function of d-luciferin dose. The time course of luciferase expression and the influence of multidosing of substrate were measured by BLI. The recovery of luciferase from the liver of hydrodynamically dosed mice allowed calibration of the BLI measurements. The results establish BLI's limit-of-detection at 20 pg of luciferase per liver following a hydrodynamic dose of 100 pg of plasmid DNA. These results demonstrate that BLI is both sensitive and linear and should allow for the direct comparison of the efficiency of gene transfer vectors that target the liver.     

Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography

Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper, we describe the use of soft X-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft X-ray tomography has the advantage of not requiring the use of any staining or fixation protocols—cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft X-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.     

The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats

Recent studies of Parkinson's disease indicate that dorsal motor nucleus of nerve vagus is one of the earliest brain areas affected by alpha-synuclein and Lewy bodies pathology. The influence of electrical stimulation of vagus nerve on elemental composition of dopamine related brain structures in rats is investigated. Synchrotron radiation based X-ray fluorescence was applied to the elemental micro-imaging and quantification in thin tissue sections. It was found that elements such as P, S, Cl, K, Ca, Fe, Cu, Zn, Se, Br and Rb are present in motor cortex, corpus striatum, nucleus accumbens, substantia nigra, ventral tectal area, and dorsal motor nucleus of vagus. The topographic analysis shows that macro-elements like P, S, Cl and K are highly concentrated within the fiber bundles of corpus striatum. In contrast the levels of trace elements like Fe and Zn are the lowest in these structures. It was found that statistically significant differences between the animals with electrical stimulation of vagus nerve and the control are observed in the left side of corpus striatum for P (p = 0.04), S (p = 0.02), Cl (p = 0.05), K (p = 0.02), Fe (p = 0.04) and Zn (p = 0.02). The mass fractions of these elements are increased in the group for which the electrical stimulation of vagus nerve was performed. Moreover, the contents of Ca (p = 0.02), Zn (p = 0.07) and Rb (p = 0.04) in substantia nigra of right hemisphere are found to be significantly lower in the group with stimulation of vagus nerve than in the control rats.     

Intracellular mapping of the distribution of metals derived from the antitumor metallocenes

The intracellular distribution of transition metals in V79 Chinese hamster lung cells treated with subtoxic doses of the organometallic anticancer complexes Cp₂MCl₂, where Cp is η ⁵ -cyclopentadienyl and M is Mo, Nb, Ti, or V, has been studied by synchrotron-based X-ray fluorescence (XRF). While significantly higher concentrations of Mo and Nb were found in treated cells compared with control cells, distinct differences in the cellular distribution of each metal were observed. Analysis of thin sections of cells was consistent with some localization of Mo in the nucleus. Studies with a noncytotoxic thiol derivative of molybdocene dichloride showed an uneven distribution of Mo in the cells. For comparison, the low levels of Ti and V in cells treated with the more toxic titanocene and vanadocene complexes, respectively, resulted in metal concentrations at the detection limit of XRF. The results agree with independent chemical studies that have concluded that the biological chemistry of each of the metallocene dihalides is unique.Electronic Supplementary Material Supplementary material is available for this article at .     

Live biospeckle laser imaging of root tissues

Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.     

The ligand environment of zinc stored in vesicles

Zinc serves regulatory functions in cells and thus, several mechanisms exist for tight control of its homeostasis. One mechanism is storage in and retrieval from vesicles, so-called zincosomes, but the chemical speciation of zincosomal zinc has remained enigmatic. Here, we determine the intravesicular zinc-coordination in isolated zincosomes in comparison to intact RAW264.7 murine macrophage cells. In elemental maps of a cell monolayer, generated by microbeam X-ray fluorescence, zincosomes were identified as spots of high zinc accumulation. A fingerprint for the binding motif obtained by μXANES (X-ray absorption near edge structure) matches the XANES from isolated vesicles; zinc is not free, but present as a complexed form (average coordination; 1.0 sulfur, 2,5 histidines 30 and 1.0 oxygen), resembling regulatory or catalytic zinc sites in proteins. Such coordination enables reversible binding, acting as a ‘zinc sink’, facilitating the accumulation of high amounts of zinc against a concentration gradient.     

Computer aided fluorescence imaging of photosynthetic systems

A fluorescence video imaging system utilizing relatively inexpensive commercial components is described. The instrument utilizes a black and white CCD video camera detector, a commercial video imaging board and a IBM-AT compatible computer. The color output of the imaging board greatly aids in the users ability to visually discriminate areas of interest in the video field. Software development that enables the user to capture kinetic traces in real time from the video images is also described. The system is used to monitor fluorescence from photosynthetic systems. The usefulness of the system in screening for photosynthetic mutants is also demonstrated. The cost of the system can be kept below $12,000.Abbreviations CCD charge-coupled device - DCMU diuron, 3-[3,4-Dichlorophenyl]1,1-dimethylurea - AGC automatic gain control - LUT look-up table - AOI area of interest - CPU central processing unit - RAM random access memory - ADC analog-to-digital converter - FVIPS fluorescence video image processing software - I/O input/output - F₀ dark-level fluorescence - OIDPSMT characteristic transient components, where O is dark level, I is intermediary peak, D is dip, P is peak of fast transient, S is quasi-steady state level, M is second maximum, T is terminal level     

Mass spectrometry imaging and profiling of single cells

Mass spectrometry imaging and profiling of individual cells and subcellular structures provide unique analytical capabilities for biological and biomedical research, including determination of the biochemical heterogeneity of cellular populations and intracellular localization of pharmaceuticals. Two mass spectrometry technologies-secondary ion mass spectrometry (SIMS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS)-are most often used in micro-bioanalytical investigations. Recent advances in ion probe technologies have increased the dynamic range and sensitivity of analyte detection by SIMS, allowing two- and three-dimensional localization of analytes in a variety of cells. SIMS operating in the mass spectrometry imaging (MSI) mode can routinely reach spatial resolutions at the submicron level; therefore, it is frequently used in studies of the chemical composition of subcellular structures. MALDI MS offers a large mass range and high sensitivity of analyte detection. It has been successfully applied in a variety of single-cell and organelle profiling studies. Innovative instrumentation such as scanning microprobe MALDI and mass microscope spectrometers enables new subcellular MSI measurements. Other approaches for MS-based chemical imaging and profiling include those based on near-field laser ablation and inductively-coupled plasma MS analysis, which offer complementary capabilities for subcellular chemical imaging and profiling.     

Non-invasive imaging of roots with high resolution X-ray micro-tomography

X-ray micro-tomography is a well-established technique for non-invasive imaging and evaluation of heterogeneous materials. An inexpensive X-ray micro-tomography system has been designed and built for the specific purposes of examining root growth and root/soil interactions. The system uses a silver target X-ray source with a focal spot diameter of 80 m, an X-ray image intensifier with a sampling aperture of about 100 m, and a sample with a diameter of 25 mm. Pre-germinated wheat and rape seeds were grown for up to 8–10 days in plastic containers in a sandy loam soil sieved to < 250 m, and imaged with the X-ray system at regular intervals. The quality of 3 D image obtained was good allowing the development and growth of both root axes and some first-order laterals to be observed. The satisfactory discrimination between soil and roots enabled measurements of root diameter (wheat values were 0.48–1.22 mm) in individual tomographic slices and, by tracking from slice to slice, root lengths were also measured. The measurements obtained were generally within 10% of those obtained from destructive samples measured manually and with a flat-bed scanner. Further developments of the system will allow more detailed examination of the root:soil interface.     

Quantitative imaging of oil storage in developing crop seeds

In this article, we present a tool which allows the rapid and non-invasive detection and quantitative visualization of lipid in living seeds at a variety of stages using frequency-selected magnetic resonance imaging. The method provides quantitative lipid maps with a resolution close to the cellular level (in-plane 31 µm × 31 µm). The reliability of the method was demonstrated using two contrasting subjects: the barley grain (monocot, 2% oil, highly compartmentalized) and the soybean grain (dicot, 20% oil, economically important oilseed). Steep gradients in local oil storage were defined at the organ- and tissue-specific scales. These gradients were closely coordinated with tissue differentiation and seed maturation, as revealed by electron microscopy and biochemical and gene expression analysis. The method can be used to elucidate similar oil accumulation processes in different tissues/organs, as well as to follow the fate of storage lipids during deposition and subsequent mobilization.     

Effects of Axotomy on Distribution and Concentration of Elements in Rat Sciatic Nerve

X-ray microprobe analysis was used to determine the effects of axotomy on distribution and concentration (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in frozen, unfixed sections of rat sciatic nerve. Elemental concentrations were measured in axoplasm, mitochondria, and myelin at 8, 16, and 48 h after transection in small-, medium-, and large-diameter fibers. In addition, elemental composition was determined in extraaxonal space (EAS) and Schwann cell cytoplasm. During the initial 16 h following transection, axoplasm of small fibers exhibited a decrease in dry weight concentrations of K and Cl, whereas Na and P increased compared to control values. Similar changes were observed in mitochondria of small axons, except for an early, large increase in Ca content. In contrast, intraaxonal compartments of larger fibers showed increased dry weight levels of K and P, with no changes in Na or Ca concentrations. Both Schwann cell cytoplasm and EAS at 8 and 16 h after injury had significant increases in Na, K, and Cl dry weight concentrations, whereas no changes, other than an increase in Ca, were observed in myelin. Regardless of fiber size, 48 h after transection, axoplasm and mitochondria displayed marked increases in Na, Cl, and Ca concentrations associated with decreased K. Also at 48 h, both Schwann cell cytoplasm and EAS had increased dry weight concentrations of Na, Cl, and K. The results of this study indicate that, in response to nerve transection, elemental content and distribution are altered according to a specific temporal pattern. This sequence of change, which occurs first in small axons, precedes the onset of Wallerian degeneration in transected nerves.