Instrumental considerations and applications of elemental bio‐imaging
- Publication Type:
- Thesis
- Issue Date:
- 2012
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Elemental Bio-Imaging (EBI) is an established application of laser ablation inductively coupled
plasma mass spectrometry (LA-ICP-MS) that determines spatial distributions of in situ trace
element concentrations in thin sections of biological tissues. This project provides an
examination of instrumental considerations relating to method development and refinement, and
the utilisation of amended methods for the analysis of element concentration
distributions in specific biological samples.
The first instrumental consideration examined improving the methods used for matrixmatched
tissue standard preparation. The main disadvantage to the previously proposed
tissue standard preparation was a decrease in the homogeneity of sectioned tissue as the
concentrations of spiked elements increased. The decrease in homogeneity was due to the
use of relatively low concentration (1000 μg mL-1) certified single element standard solutions.
Substituting the low concentration solutions for higher concentration solutions, prepared
using soluble metal salts, allowed for smaller quantities of solution to be spiked into the
tissue. This increased homogeneity and the ability to spike a greater number of analytes into
each tissue standard.
The appropriateness of increasing laser scan speed for the reduction of total experimental
analysis time was also examined. EBI experiments normally employ scan speeds where the
distance traversed in one second is equal to or less than the diameter of the laser beam.
Consequently, data for a higher‐resolution (pixel size = 15 μm x 15 μm) image of a 5 mm x
5 mm tissue section can take upwards of 30 hours to acquire. Appropriate laser scan speeds
may be calculated by consideration of the relationship between laser scan speed, laser spot
diameter and the total scan cycle of the quadrupole mass analyser. A simple method to
calculate the laser scan speeds capable of reducing the acquisition time by up to a factor of
five whilst maintaining dimensional integrity of the image is presented in this thesis.
Two applications were developed utilising increased laser scan speeds. Both applications
were related to the study of the neurodegenerative disorder, Alzheimer’s disease, and
examined particular variables and their effects on the distribution of metals in the brain. The
first application examined the effectiveness of a new drug, PBT-2, on the redistribution of
elements in mouse brains with the zinc transporter-3 gene removed. Results indicated PBT‐2
had little effect on the distribution of 66Zn, 63Cu and 56Fe in the midbrain. The second
application examined the effect of intermittent hypoxia and a diet of advanced glycation
end‐products on element distribution in the brain. The most intriguing results were seen in
the images for 59Co. A 100-fold elevation in the concentrations of 59Co was observed
between mice exposed to intermittent hypoxia, those in a control environment and wild-type
mice not exposed to the experiment’s settings.
An examination into the use of H2 as a reaction gas was conducted. The improvement of the
analytical performance of imaging experiments was considered for a range of masses with
spectral interferences, including the 40Ar16O+ spectral interference on 56Fe+. At low
(< 1.0 mL min-1) H2 flow rates, greater spectral interference due to H+ adducts were observed
for 57Fe than with the reaction mode off. At higher flow rates, up to 3.0 mL H2 per minute, the
spectral interferences were reduced leading to an improvement in the limits of analysis for
masses with O- and N-based polyatomic interferences.
EBI is typically performed using spatial resolutions of 30 μm x 30 μm and above. Higher
resolution imaging is desirable for many biological applications. As a culmination of the
project, the combination of the use of improved matrix‐matched tissue standards with the
use of the reaction cell and increased image acquisition speeds was employed. The
combination of improved parameters resulted in a high quality image of the area of the brain
including the substantia nigra being prepared at a resolution of 6 μm x 6 μm for 56Fe.
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