Absolute quantitative electron microscopy of thin biological specimens by energy-dispersive x-ray microanalysis and densitometric mass determination

Linders, P.W.J.; Vorstenbosch, R.A. van de; Smits, H.T.J.; Stols, A.L.H.; Stadhouders, A. M.

doi:10.1016/s0003-2670(00)84508-0

Cited by 6 publications

(4 citation statements)

References 20 publications

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“…Procedures for mass determination by measurement of the transmission of the total electron beam have been described in detail by Halloran et al (1978), Pozsgai 8z Barna (1983) and Linders et al (1982Linders et al ( ,1984. Similar procedures based on measurement of the attenuation of the zero-loss peak in the electron energy-loss spectrum have been described by Leapman et al (1984) and Hosoi et al (198 1).…”

Section: Resultsmentioning

confidence: 99%

Properties of frozen sections relevant to quantitative microanalysis

Hall¹

1986

Journal of Microscopy

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SUMMARY Difficulties in the quantitative X‐ray microanalysis of frozen sections may conceivably arise from ice‐crystal damage and from electron‐beam damage. X‐ray peak‐to‐continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice‐crystal formation existing prior to sublimation. The experiments in these reports are re‐interpreted here; it is argued that peak intensities may be affected by ice‐crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak‐to‐continuum method is affected by beam‐induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold‐stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen‐hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen‐hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X‐ray analysis at very high resolution.

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Section: Resultsmentioning

confidence: 99%

Properties of frozen sections relevant to quantitative microanalysis

Hall¹

1986

Journal of Microscopy

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“…It is we11 known that in electron microscopy the contrast is related to the mass distribution in the specimen. Originally, the mass thickness was determined from the attenuation of the electron beam after passing the specimen according to Beer's law Burge and Silvester, 1959;Halloran et al, 1978;Linders et al, 1982Linders et al, , 1984Marton and Schiff, 1941;Poszgai and Barna, 1983;S i k h , 1983;Wall, 1979;Bahr, 1962, 1965). However, in conventional electron microscopy and also in STEM the unscattered electrons that have not interacted with the specimen and consequently do not contribute to the information concerning the mass thickness are superimposed on the brightfield signal.…”

Section: Mass Determination In Freeze-dried Cryosectwnsmentioning

confidence: 99%

“…This might be due to the effect of extraneous X-rays on the background intensity measurements as discussed above. Linders et al (1982Linders et al ( ,1984 have compared X-ray microanalysis and measurements of the electron beam current after attenuation by the specimen for mass determination and found higher accuracy in the electron beam current measurements.…”

Section: Mass Determination In Freeze-dried Cryosectwnsmentioning

confidence: 99%

X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections

Zierold

1988

J. Elec. Microsc. Tech.

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The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-micron thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.

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“…Quantitative X-ray microanalysis in an electron probe instrument CTEM, STEM, or SEMI requires that either the beam current be measured accurately or that it be kept constant accurately (Linders et al, 1984). For Morgan and Davies (1982) it was necessary to know the beam current in order to ascertain that the electron dose was sufficiently low not to cause loss of elements such as S, C1, K, Na, and P by volatilization.…”

Section: Introductionmentioning

confidence: 99%

Modification of a TEM‐goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X‐ray microanalysis

Stols

Smits

Stadhouders

1986

J. Elec. Microsc. Tech.

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KEY WORDS STEM specimen holder, Beam current, X-ray microanalysis, Transmission electron microscopy ABSTRACTIn order to have available a specimen holder suited to measure the beam current as is often required in quantitative electron probe X-ray microanalysis, the rod of a low background beryllium specimen holder of a transmission electron microscope was modified. The tip was electrically insulated from the mass of the microscope and connected electrically to the central contact of a BNC connector mounted on the specimen holder handle. With this modified specimen holder the current absorbed by the specimen andor the specimen holder could be measured easily and accurately. The modified specimen holder has been used to measure the beam current stability of an analytical electron microscope under various conditions. Data were obtained for tungsten as well as lanthanum hexaboride cathodes. Small changes to other types of specimen tips made it possible to exchange these for the low background tip.

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Absolute quantitative electron microscopy of thin biological specimens by energy-dispersive x-ray microanalysis and densitometric mass determination

Cited by 6 publications

References 20 publications

Properties of frozen sections relevant to quantitative microanalysis

Properties of frozen sections relevant to quantitative microanalysis

X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections

Modification of a TEM‐goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X‐ray microanalysis

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