In-vacuum micro-PIXE analysis of biological specimens in frozen-hydrated state

https://doi.org/10.1016/j.nimb.2007.02.017Get rights and content

Abstract

The first in-vacuum measurements of biological specimens in frozen-hydrated state using proton microbeam have been performed at Materials Research Group, iThemba LABS, South Africa. A commercially available cryotransfer system used in electron microscopy has been adapted for this purpose. The analyzed material was frozen in propane cooled by liquid nitrogen, fractured, carbon coated and transferred onto the cold stage (100 K) in the nuclear microprobe chamber. Micro-PIXE and simultaneous proton backscattering was performed using 3 MeV proton beam. Monitoring of water vapour composition during the proton bombardment showed good stability of the analyzed material. Quantitative results were obtained by standardless method, tested using 20% gelatine standards with added PbCl2. The differences between the concentrations obtained for frozen-hydrated and next freeze-dried specimens and the value calculated on the basis of weighted mass of PbCl2 added to gelatine were statistically insignificant. Leaf tissue of Senecio anomalochrous and larvae of Chysolina pardalina were used as examples of plant and animal tissue. Quantitative elemental mapping of frozen-hydrated specimens compared with subsequent analysis of the same areas after freeze-drying revealed the same distribution pattern in both cases.

Introduction

Studies of elemental distribution in soft biological material are only meaningful when they reflect the true location and concentration of elements in living tissues and cells. The advantage of using frozen-hydrated specimens lies in the fact that the analyzed material remains at its full water content and volume and tissue cells retain the same relation to each other as they did in their life [1], [2]. Analyses at low temperature are also useful in studies of non-biological specimens, especially volatile, hygroscopic or susceptible to electron or proton beam.

The first X-ray microanalyses of biological specimens in frozen-hydrated state date back to the early 1970s and were performed with electron excitation of characteristic X-rays [3], [4]. Fast progress in this direction led to the development of preparation and handling procedures of analysis of frozen-hydrated specimens for scanning and transmission electron microscopes. Limits and restrictions of quantitative elemental analysis have been established [3], [5], [6] and eventually the equipment necessary for such analyses became available commercially. Electron microscopes (SEM or TEM) are equipped with a specimen support cooled to liquid nitrogen temperature (cold stage), a cryopreparation or cryotransfer chamber, which is directly attached to the specimen chamber of the microscope column and a source of low temperature. The main function of the cryopreparation chamber is to keep specimens at low temperature (below 120 K) during preparation procedures − freeze fracturing, sputtering or evaporation. Integrated with a microscope, such chamber enables transfer of cryofixed specimens to the microscope under vacuum.

Despite long tradition of analysis of dry biological specimens by PIXE/micro-PIXE [7], [8], [9], [10], [11] there has been surprisingly low interest in conducting analyses of tissues and cells in frozen-hydrated state. Only two attempts to measure cooled biological specimens can be noted throughout the whole period of the development and use of proton microprobes. Horowitz et al. [12] were the first to use a simple cold stage cooled to 243 K by circulating liquid nitrogen and the final temperature of specimens was stabilized by thermistor-controlled heating element. This temperature was, according to their claims, “sufficient to immobilize diffusible substances on the scale of resolution employed in these experiments”. However, the samples were not kept in vacuum but in air, enclosed in the special chamber equipped with 7 μm Kapton window upon which a stream of dry nitrogen was blown. The technique has been demonstrated on specimens of rat eye and kidney tissue. Maps of elemental distribution were obtained for a proton beam of 100–150 μm in diameter, 2 MeV energy and 5 nA current. This was the first proof that PIXE could easily overcome the main problem encountered when using electrons for creation of elemental maps, where intense generation of continuum radiation strongly decreases peak-to-background (P/B) ratio [13]. More recently Sakai et al. [14] modified the nuclear microprobe system to analyze biological specimens at low temperature in air by introducing the CryoJet® (Oxford Instruments) cooling device blowing the cryogenic gas (N2) on the sample. According to their claims, the specimen temperature was maintained at 100 K during analysis. Elemental maps of onion layer peeled and set onto Mylar foil were obtained using 3 MeV protons and 100 pA beam current. Both experiments did not reveal specimen damage due to proton beam bombardment.

Despite perceived need for “in vacuum” analyses at low temperature [10] there seems to be no earlier systematic attempts in this direction. Our aim was to perform low temperature (below 100 K), quantitative measurements of elemental composition and distribution in frozen-hydrated biological materials by micro-PIXE in vacuum, at lateral resolutions comparable with these routinely achieved when analyzing freeze-dried specimens.

Section snippets

Adaptation of the cryotransfer system

The commercially available cryotransfer system (E7400 Cryotrans System manufactured by Bio-Rad Microscience Division, USA) has been attached to the experimental chamber of the nuclear microprobe (NMP) at Materials Research Group, iThemba LABS, South Africa, after necessary adaptation [15]. This NMP chamber is the octagonal chamber manufactured by Oxford Microbeams [16], with modified top and computer-controlled specimen positioning system using stepper motors. For routine measurements of

Standards

Handling of cold specimens between cryogen and the cryopreparation chamber is crucial for a final state of investigated material, especially when it cannot be fractured or sectioned in the cryopreparation chamber before analysis. Frost deposition (mass gain) and specimen melting could change its life-like structure and elemental composition or might introduce artefacts in the quantification procedure, thus influencing the final results. A balance between the rate of nitrogen flow through the

Conclusions

Quantitative elemental analyses of samples in frozen-hydrated state using micro-PIXE with complementary information on content of water and light elements forming biological matrix obtained from simultaneously performed proton backscattering are possible with minimum detection limits down to 1 μg g−1 for proton beam current not exceeding 150 pA, with samples kept at temperature below 110 K during all operation and measurements. Comparison of results obtained for frozen-hydrated material can be used

Acknowledgements

We are greatly indebted to Rosemary White from Microscopy Centre of CSIRO Plant Industry in Canberra, Australia, for kind donation of their E7400 Cryotrans System. We further acknowledge technical help and suggestions of Karl Springhorn, Lawrence Ashworth, Chris Theron and Rob McAlister. Part of financial support for this project was provided by the International Atomic Energy Agency.

References (27)

  • L.R. Doolittle

    A semiautomatic algorithm for Rutherford backscattering analysis

    Nucl. Instr. and Meth. B

    (1986)
  • R. Amirikas et al.

    Measurement of (p,p) elastic cross sections for C, O and Si in the energy range 1.0–3.5 MeV

    Nucl. Instr. and Meth. B

    (1993)
  • A.J. Saubermann et al.

    The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and X-ray microanalysis

    J. Microsc.

    (1975)

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    1

    On leave from the Faculty of Physics and Applied Computer Science Informatics, AGH University of Science and Technology, 30-059 Kraków, Poland.

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