Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

Abstract

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides three-dimensional distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L 2,3 edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.

On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography

“…Recently, we have reported a similar quantification scheme based on scattering theory and on a calibration of the experimental parameters of the microscope to determine in absolute terms the mass of biological macromolecules directly from their STEM signal (Sousa and Leapman, 2007). The present results further support the validity of image simulations based on scattering theory for assessing the accuracy of quantitative dark-field STEM.…”

“…Therefore it was straightforward to remove the fiducial features by substituting their pixel intensities with the mean pixel intensity of the entire image. Tomographic reconstruction of the aligned tilt series was performed using the simultaneous iterative reconstruction technique (SIRT) algorithm (Gilbert, 1972;Herman, 1980), which we have implemented in our group (Aronova et al, 2007). The specimen of Nanogold embedded in 100 nm of carbon was also reconstructed with weighted back-projection (WBP) (Radermacher, 2006) in IMOD (Kremer, 1996).…”

“…Because the model tomogram does not match all the noise variations in the tilt images, some level of smoothing takes place. In SIRT, the optimum number of iterations towards the best solution depends on the original amount of noise in the images, and this subject is discussed in more details elsewhere (Aronova et al, 2007). 4.6.…”

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM

“…We acquired a STEM tomographic tilt series from this cell and performed reconstruction with SIRT. As demonstrated by Aronova et al (2007), reconstruction with SIRT enables generation of tomograms whose projected voxel intensities match the pixel intensities of the original tilt images. Quantitative information is therefore retained within such tomograms.…”

Die Renaissance der Elektronenenergieverlust‐Spektroskopie

“…[2] Idealerweise konnte man so nicht nur die Zusammensetzung und Dicke (anhand der Position und abgeschwächten Signale der Verlustpeaks) der untersuchten Proben ermitteln. Wie an anderer Stelle beschrieben, [2][3][4] [7] zeigen, dass 1) die Gesamtverteilung von Biomolekülen -Proteine und Nucleinsäuren eingeschlossen -aus der Intensität des EELS-Signals für die KSchale von Stickstoff erhalten wird, 2) Proteine mit einem hohen Gehalt an den Aminosäuren Cystein und Methionin durch das EELS-Signal von Schwefel detektiert werden können und 3) die Verteilung von Nucleinsäuren, phosphorylierten Proteinen und Phospholipiden aus den EELSSignalen für Phosphor hervorgeht.…”