RESUMEN
Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.
Asunto(s)
Fenómenos Fisiológicos Celulares , Microanálisis por Sonda Electrónica/métodos , Animales , Calcio/análisis , Células/ultraestructura , Cloro/análisis , Gráficos por Computador , Criopreservación , Bases de Datos Factuales , Microanálisis por Sonda Electrónica/instrumentación , Liofilización , Secciones por Congelación , Humanos , Procesamiento de Imagen Asistido por Computador , Glomérulos Renales/química , Glomérulos Renales/citología , Glomérulos Renales/ultraestructura , Túbulos Renales Proximales/química , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/ultraestructura , Leishmania tropica/química , Leishmania tropica/citología , Leishmania tropica/ultraestructura , Magnesio/análisis , Microcomputadores , Microscopía Electrónica , Mitocondrias/química , Mitocondrias/ultraestructura , Músculos/química , Músculos/citología , Músculos/ultraestructura , Miocardio/química , Miocardio/citología , Miocardio/ultraestructura , Mucosa Nasal/química , Mucosa Nasal/citología , Mucosa Nasal/ultraestructura , Fósforo/análisis , Potasio/análisis , Sodio/análisis , Programas InformáticosRESUMEN
We monitored the mass thickness of egg yolk phosphatidylcholine multilayers at several temperatures during electron irradiation. The rate of irradiation-induced mass loss was reduced substantially when this specimen was cooled to liquid nitrogen temperature from room temperature. Additional cooling to liquid helium temperature caused an additional reduction of mass-loss rate. The characteristic doses D(1/e), which are the slopes of the logarithm of the differential mass thickness against dose, were approximately 7 x 10(3) e/nm2 at 290 K, 8 x 10(4) e/nm2 at 130 K, and 1.4 x 10(5) e/nm2 at less than 10 K. The fractions of the original mass thickness that remained after arbitrarily high doses were about 69% at 290 K, 72% at 130 K, and 77% at less than 10 K.
Asunto(s)
Microscopía Electrónica/normas , Fosfatidilcolinas/efectos de la radiación , Animales , Yema de Huevo , Electrones , Fosfatidilcolinas/química , TemperaturaRESUMEN
Because a long time is generally required to generate X-ray maps of specific elements by electron beam methods, images are subject to a loss of resolution due to stage movement. Methods have been previously described for correcting stage drift during exposure by sensing the drift and deflecting the beam to follow the stage; but these methods require modifications of the equipment. When the drift is not excessive, it is possible to correct a series of images after the exposure series is finished. Here we demonstrate two methods for correcting the drift, one based on manual assignment of specimen position and one on the use of cross-correlation functions to determine objectively the misalignment of images in the series. The success of the methods is illustrated in calcium-specific images of a bone section that show the collagen periodicity after drift correction.
Asunto(s)
Microanálisis por Sonda Electrónica/métodos , Huesos/ultraestructura , Colágeno/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Movimiento (Física)RESUMEN
A previous measurement showed that mass loss from collodion supported by thin carbon films was linear with electron exposure at liquid helium temperature. No other organic solid had shown a linear loss of mass at any temperature. When measurements of collodion were done using titanium supports, the loss of mass proceeded exponentially with exposure at liquid helium temperature. This result suggested that the differing electrical conductivities of these substrates might be the cause of the different mass loss effects. Carbon films, which are typically used at ambient temperatures, have much lower electrical conductivity at very low temperature than titanium films. This suggested that specimen preparation materials and techniques used routinely for room temperature studies may need to be modified when microscopy is done using superconducting objective lenses. For both substrates, the rate of mass loss is slowest at liquid helium temperature.
Asunto(s)
Frío , Colodión , Microscopía Electrónica/métodos , Carbono , Conductividad Eléctrica , Helio , TitanioRESUMEN
Water can be a substantial proportion of the residual gas in modern electron microscopes even when frozen hydrated specimens are not used. During measurements of the mass thickness of thin collodion film specimens at low temperatures, it was found that a volatile surface layer (condensed water) modified the apparent rate of mass loss induced by radiation exposure. Mass loss can be enhanced by the presence of water (specimen "etching"), or mass loss can be masked by the dynamic adsorption of water to the specimen surface. The microscope or the grid can be a secondary source of the water; even with cold anticontaminator plates in the vicinity of the specimen, water can be desorbed by x-rays or backscattered electrons. In one typical situation, the mass loss rate appears reduced (due to water adsorption), but the ultimate damage is greater (due to etching). These results illustrate that care must be taken in interpreting mass thickness measurements made in the presence of water and that the lowest stage temperature does not necessarily produce the best observation conditions for all specimens.
Asunto(s)
Microscopía Electrónica , Agua , CongelaciónRESUMEN
A method is described for using a general purpose multi-process minicomputer for the real-time display of graphic summaries of a variety of spectroscopic results while the spectrum acquisition is simultaneously underway.
Asunto(s)
Gráficos por Computador , Microanálisis por Sonda Electrónica/métodos , Programas InformáticosRESUMEN
The mass thickness of collodion films has been monitored at several temperatures, under conditions typical of electron microscopy, through the use of an electron energy loss spectrometer. Compared to room temperature, only a five-fold reduction in the rate of mass loss was observed through the use of a commercial liquid nitrogen cooled stage; in contrast, the rate of mass loss was reduced more than one hundred fold when these films were held at liquid helium temperature.