Low energy loss electron microscopy of chromophores

A novel prism-mirror-prism imaging electron spectrometer with 1 eV energy resolution for a transmission electron microscope permits imaging with spectral energies corresponding to light-optical colour absorptions. The instrument selects the molecular orbital excitations of natural chromophores or of specific dyes normally used in biological light microscopy for delineation and chemical identification, but images them with electron microscopic detail. Heavy atom contrast agents customarily used in electron microscopy are not required. The first results exploit the intrinsic red colour of hematin molecules to demonstrate the potential of the technique and address its spatial resolution. Glycosaminoglycans in cartilage stained with Alcian blue are selectively depicted in situ by means of the electron-induced molecular absorption of this chromophore. Thus, with the use of specific colours the direct or indirect analysis of local chemistry by electron microscopy is possible, and can be carried out with a depiction of spatial detail as small as 16 A, or at least 100-fold finer than observed by light microscopy.     

Characterization of palladium hydride films by electron energy loss spectroscopy and electron diffraction

Electrolytic charging is used to hydrogenate Pd films to full stoichiometry PdH, which is checked by making use of the relation between the lattice constant of the Pd-H system and its hydrogen content. The role of the surface barrier in preventing hydrogen escape from fully charged specimens is studied with EELS technique. Electron energy loss spectra collected at different collector aperture deviation angles in a STEM are used to separate the volume loss component from the surface loss component. The results are compared with those of Pd metal and results of PdH obtained with other techniques.     

Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging

Glucose utilization of four cerebral cortex and 35 subcortical regions (CGU) was analyzed in three models of cholinergic seizures induced by the following compounds: 1) soman (pinacolylmethylphosphonofluoridate) an organophosphorus cholinesterase inhibitor, 100 microg/kg SC after pretreatment with pyridostigmine 26 microg/kg IM (n = 6); 2) physostigmine, a carbamate cholinesterase inhibitor, 1.31 mg/kg infused IV over 75 min (n = 6); and 3) pilocarpine, a direct cholinergic agonist, 30 mg/kg SC (n = 6). Physostigmine and pilocarpine were preceded by 3 mmol/kg LiCl IP 20 hrs earlier. Animals injected with saline SC (n = 6) were used as controls. Step-wise discriminant analysis successfully classified 100% of the cases into the four experimental groups with data from only six regions. Pyridostigmine-soman induced the most widespread and greatest increases in CGU. More restricted and lower levels of activation were observed with Li-pilocarpine while Li-physostigmine induced significant increases in CGU only in globus pallidus, entopeduncular nucleus, and substantia nigra. These three regions, which are functionally related, were also activated in the other two models of cholinergic convulsions and may represent the initial step in cholinergic activation of the CNS. Li-pilocarpine failed to activate most of the brainstem and the superior colliculus. All cortical regions were activated by Li-pilocarpine and pyridostigmine-soman, while they were inhibited by Li-physostigmine. This phenomenon may be due in part to the lack of activation with physostigmine of the basal forebrain nuclei (lateral septum, medial septum, vertical and horizontal limbs of the diagonal band, and substantia innominata) resulting in a decreased drive of cortical metabolism.     

High-resolution imaging of protein phosphorylation in squid axons and synapse by electron energy loss spectroscopy

We demonstrate that clusters of phosphorus atoms can be detected in energy loss spectroscopic images (ESI) of cytoskeletal proteins of squid axons. In series of images taken at four energy windows below and three windows above the phosphorus P-L2,3 ionization edge, signal-to-background intensity differences were analyzed by videodensitometry. A distinct increase of relative intensities was recorded above the phosphorus edge in neurofilaments of the peripheral giant axon and in those of the presynaptic terminal. A high level of neurofilament phosphorylation in the peripheral axon supports previous biochemical and immunochemical results, but our finding of phosphorylated neurofilaments in the presynaptic axon conflicts with these studies. Our method may be advantageous for analysis with high elemental and spatial resolution of the phosphorylation state of cytoskeletal protein molecules in situ.     

Characterisation of niobium and vanadium hydrides by electron energy loss spectroscopy and by STEM

The transmission electron energy loss spectra from hydrogen-containing vanadium and niobium are compared with those from the pure metals. The hydrogen induces an additional small energy loss peak at very low energies, well below that due to the plasmon loss. This peak can be interpreted as due to a band of states induced by the hydrogen several eV below the conduction bands of the metal. Because these experiments are performed in a STEM, it is possible to verify the structure of the hydride by electron microscopy and diffraction and to be certain that the energy-loss spectra come from well characterised material.     

Optimization of 3D conformal electron beam therapy in inhomogeneous media by concomitant fluence and energy modulation

The possibilities of using simultaneous fluence and energy modulation techniques in electron beam therapy to shape the dose distribution and almost eliminate the influences of tissue inhomogeneities have been investigated. By using a radiobiologically based optimization algorithm the radiobiological properties of the tissues can be taken into account when trying to find the best possible dose delivery. First water phantoms with differently shaped surfaces were used to study the effect of surface irregularities. We also studied water phantoms with internal inhomogeneities consisting of air or cortical bone. It was possible to improve substantially the dose distribution by fluence modulation in these cases. In addition to the fluence modulation the most suitable single electron energy in each case was also determined. Finally, the simultaneous use of several preselected electron beam energies was also tested, each with an individually optimized fluence profile. One to six electron energies were used, resulting in a slow improvement in complication-free cure with increasing number of beam energies. To apply these techniques to a more clinically relevant situation a post-operative breast cancer patient was studied. For simplicity this patient was treated with only one anterior beam portal to clearly illustrate the effect of inhomogeneities like bone and lung on the dose distribution. It is shown that by using fluence modulation the influence of dose inhomogeneities can be significantly reduced. When two or more electron beam energies with individually optimized fluence profiles are used the dose conformality to the internal target volume is further increased, particularly for targets with complex shapes.     

Drug targeting and metabolic investigations of cryoprepared tumor cells with analytical electron energy loss spectroscopy

Analytical electron microscopy is an ideal tool for holistic data acquisition on biological systems. The use of analytical electron microscopy for both, the investigation of micropharmacokinetic problems and metabolic studies, is becoming more and more important. Depending on the mode of investigation, it is possible to localize drugs and xenobiotics precisely in situ under optical control or to quantify their uptake and distribution in the corresponding target cells without disintegrating the cell or tissue material. In this paper, we present instructive examples for the application of analytical electron energy loss spectroscopy in transmission electron microscopy in order to investigate the cellular uptake and distribution of cisplatin and cyclophosphamide and the metabolic changes induced by an alteration in the extracellular calcium concentration in a holistic manner.     

采用电子能量损失谱成像技术对富铬钢的沉淀物进行综合分析

本文通过采用先进的分析透射电子显微镜方法,阐述了硼合金为9%的铬钢的热处理过程和蠕变测试过程中的微结构发展,着重强调了硼的作用。通过透射电子显微镜扫描模式获得的电子能量损失谱图像提供了完整的元素信息,这些信息通过高空间和高能量分辨率的碳,氮,铁,铬,钒的结构量化图（单位为atoms/nm³）进行传达。研究证实了嵌在M₂₃(C,B)₆颗粒中的MX沉淀物,并分析了其硼含量。为了识别氮化钒颗粒从早期阶段转变到复杂的氮化改性Z相,通过应用二元直方图分析对能源过滤透射电子显微镜（TEM）的元素图进行处理。另外,该研究已经绘制了蠕变检测样本的原奥氏体晶界图,并通过能源过滤透射电子显微镜,电子能量损失和X射线光谱图识别了相图。
     

Spectral diffusion and the energy landscape of a protein

We present a novel type of spectral diffusion experiment in the millikelvin range to characterize the energy landscape of a protein as compared with that of a glass. We measure the time evolution of spectral holes for more than 300 hr after well-defined initial nonequilibrium conditions. We show that the model of noninteracting two-level systems can describe spectral diffusion in the glass, but fails for the protein. Our results further demonstrate that randomness in the energy landscape of a protein shows features of organization. There are "deep minimum" states separated by barriers, the heights of which we are able to estimate. The energy landscape of a glass is featureless by comparison.