X‐ray microanalysis of biological material in the frozen‐hydrated state by PIXE

Abstract

Elemental microanalysis of biological material in the frozen-hydrated state using in-vacuum proton induced X-ray emission is described for the first time. For this purpose, a commercially available cryotransfer system was modified and coupled to the experimental chamber of the nuclear microprobe (NMP). The analyzed material was frozen in propane cooled by liquid nitrogen, fractured, carbon coated, and transferred onto the cold stage (100 K) of the nuclear microprobe chamber. Micro-PIXE and simultaneous proton backscattering was performed using a 3 MeV proton beam. Quantitative results were obtained by the standardless method, and tested using 20% gelatin standards. Monitoring of the gas composition inside the system by means of mass spectrometry performed before, during, and after proton bombardment showed good stability of the analyzed material for proton currents not exceeding 150 pA. Average concentrations of light elements (C, N, O, and indirectly H) were also obtained by the proton backscattering technique. No losses of elements measurable by particle-induced X-ray emission (PIXE) during proton irradiation were found during repetitive, short analyses of the same micro areas of gelatin standards. Measurements of thick sections of selected plant and animal material in the frozen-hydrated state-leaf sections of the plant Senecio anomalochrous Hilliard (Asteraceae) and larvae of Chysolina pardalina Fabricius (Chrysomelidae)-showed very good preservation of morphology and elemental distribution. Limits of detection of the order of a few micro g g(-1) were obtained for most elements.

Elemental distribution by cryo-micro-PIXE in the zinc and cadmium hyperaccumulator Sedum plumbizincicola grown naturally

“…At site 1 the plant shoots accumulated up to 1,470 and 14,600 mg kg −1 Cd and Zn, respectively, which are similar to site 4 but higher than sites 2 and 3 (Table 4). Notwithstanding t h e v a r i a t i o n i n a c c u m u l a t i n g a b i l i t y o f S. plumbizincicola at the four sites, the metal concentrations in the plant shoots were much higher than 100 mg kg −1 for Cd and near/over 10,000 mg kg −1 for Zn, the levels defining Cd and Zn hyperaccumulating ability (van der Ent et al 2013). Moreover, the plant Cd/Zn bioconcentration factors (BCF, the ratio of the metal concentration in shoots to the concentration in soils) were > 1, especially at site 4 where BCF values were up to 40.0 and 6.52 for Cd and Zn, respectively.…”

Synchrotron-based techniques for plant and soil science: opportunities, challenges and future perspectives

“…Several studies have in fact pointed out how the distribution and speciation of various elements can be altered when, for example biological samples, are freeze-dried (e.g. Tylko et al 2007). For this reason in vivo approaches or the use of frozen hydrated biological samples are generally preferred.…”

“…A few authors have covered related subjects and in particular we would to acknowledge the excellent earlier reviews by Tuniz et al (1991), Sutton et al (1994) and Schulze and Bertsch (1995) and the more recent articles by Salt et al (2002), Manceau et al (2002), Gardea-Torresdey et al (2005a) and Thieme et al (2007) that have served as a starting point for this review.…”

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

“…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.…”