Biological X‐Ray Microanalysis

Abstract

By means of X-ray microanalysis it is now practical to detect approximately 10(-19) g of an element in a static-probe analysis within an ultrathin section, with analytical spatial resolution in the range 20--30 nm. The main difficulties for biological microanalysis are connected not with sensitivity but with specimen preparation and beam damage. Careful cryopreparation, beginning with the quench-freezing of a small block of tissue, is essential even for determining the storage sites, or sites of binding in vivo, of physiologically active elements. In frozen-dried or frozen-hydrated sections of quench-frozen tissue, it is now possible to measure local mass fractions of diffusible as well as of bound elements.

Sub-second quenched-flow/X-ray microanalysis shows rapid Ca2+ mobilization from cortical stores paralleled by Ca2+ influx during synchronous exocytosis in Paramecium cells

“…In addition, we controlled [Ca] in the Ca bead standards by atomic absorption or neutron activation (data not shown). As confirmed by these two independent physical methods, the spot mode analysis allowed us the calibration of the Ca Ka counts and a direct indication of [Ca] values per unit volume (rather than per kg dry weight) of subcellular structure (Chandler, 1976;Dörge et al, 1978;Hall, 1979). Effects of osmium (mainly fluorescence), though small (< 25%), were corrected based on an experimentally determined correction factor (Hardt and Plattner, 1999).…”

Total and free myoplasmic calcium during a contraction cycle: x‐ray microanalysis in guinea‐pig ventricular myocytes.

“…The ratio between characteristic and continuum counts is linearly related to the concentration (mass fraction) of an element in the analysed volume (Hall, 1979). The 'Quantem' program of the Link analytical system is based on the characteristic continuum theory of Hall (1979), and was used to calculate the concentrations of the elements with atomic number > 11 (Na) in mmol (kg dry weight)-'. To obtain the number of counts in a given peak, the program compares a multiple leastsquares fit of the filtered spectrum to a library of primary reference files containing pure element spectra.…”

Iron Depletion in the Remnant Kidney

“…The precise local ization of iron by EDS avoids potential inaccuracies arising due to contamination by blood or iron within cytochromes o f mitochondria. Quantification of iron was determined by the Hall continuum meth od [27], Results are expressed as mean lysosomal iron (wt% = mg% = 1.7 mmol Fe/kg tissue) or numbers of iron-containing Iysosomes per tubule, or per kidney as appropriate. Tubular damage was assessed simultaneously with EDS on unstained tissue, by a semiquantitative scale (0.0-2.0) based on the extent of damage to microvilli and dis ruption of the tubular cell, as previously described [19].…”