Real‐time quantitative elemental analysis and mapping: microchemical imaging in cell physiology

Abstract

K E Y w ORDS. X-ray microanalysis, microchemical microscopy, biological cryosections, electron microscopy, X-ray mapping, cell physiology, computer imaging. S U M M A R YRecent 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 frozenhydrated sections can be acquired to obtain element and water content from the same intracellular regions. T h e 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. structurefunction 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 uiuo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.

Saccharomyces cerevisiae Vacuole in Zinc Storage and Intracellular Zinc Distribution

“…925; Gatan, Inc., Pleasanton, CA), a scanning device, additional hard X-ray aperture, a collimated 30-mm 2 Si(Li) energy-dispersive X-ray detector (Oxford Instruments America, Palo Alto, CA), and a pulse processor; the scanning and multichannel analyses were conducted with an X-ray pulse processor (4pi Analysis spectral engine; 4pi Analysis, Inc., Durham, NC). Operating parameters and strategies for obtaining quantitative X-ray images were implemented as described extensively elsewhere (19,(25)(26)(27). Basically, 128-by 128-pixel spectral data sets were obtained at approximately ϫ10,000 magnification (1 pixel ϭ 0.2 m 2 ) with a beam current of approximately 1 nA and a dwell time of 4 s per pixel, with automation (19) and quantitation (27) carried out as previously described.…”

The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ

“…The X-ray imaging technique avoids any problems of non-uniform sublimation of ice from the sample and also provides an unbiased sampling method (LeFurgey et al, 1992). Although this technique avoids the need for experience and great care in the etching process, it has the disadvantage of the long times required to obtain the X-ray images, thus making any study requiring multiple sampling expensive for those who must pay for microscope time.…”

Electron and ion microprobe analysis of calcium distribution and transport in coral tissues

“…Quantitative numerical data, based upon the Hall peak/continuum model (Hall and Gupta, 1979), were extracted directly from the elemental maps by selecting areas of interest. Individual spectra for each pixel in the selected regions were summed and processed to yield concentrations for every element (LeFurgey et al, 1992). Elemental concentrations are given in mmol·kg -1 embedded tissue.…”