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LA-ICP-MS finds widespread use in the analysis of biological samples and its importance as an analysitical method is still increasing. Often tissues or single cells are ablated in imaging mode on glass slides, but the analysis time increases by reducing the spot size of the laser. Additionally, with increased lateral resolution a loss in sensitivity is inherently connected and this holds true in particular for single cell analysis in imaging mode. However, if only the composition of elements in a single cell needs to be measured, complete ablation of cells by single shots looks promising. This approach was already discussed by Managh et al. and will be discussed in more detail in this presentation. The aim of this study is to develop a calibration strategy for single cell analysis based on the total consumption concept. It will be shown that this concept provides quantitative results at single cell level, if the target material is ablated. We compare two different laser spot sizes, 30 µm and 110 µm. The smaller spot size of 30 µm is used for imaging and the larger spot size of 110 µm is used for the targeted ablation of complete single cells. Cells were stained by two novel cell dyes, mDOTA-Tb (CheMatech, Dijon, France), which binds to free thiol groups of proteins, and Ir-DNA intercalator (Maxpar®, Fluidigm, San Francisco, USA), which stains DNA quantitatively. Finally, quantification of Ir and Tb will be performed by matrix matching calibration, following the approach of Drescher et al.[1], i.e. spotting of a dilution series of Ir and Tb standards on nitrocellulose membrane. Results obtained so far show a good correlation between imaging and single spot ablation, indicating that the total consumption concept may serve as an acceptable assumption.
Quantification of metals in single cells by LA-ICP-MS comparison of single spot analysis and imaging
(2018)
LA-ICP-MS is increasingly used for single cell analysis in two different detection modes using either the imaging mode with subcellular resolution or alternatively single spot analysis of cells with a larger laser spot size. This study compares the analytical figures of merit of both detection modes (signal to noise, precision, accuracy, throughput), as well as ease of operation and data evaluation. Adherent 3T3 fibroblast cells were stained with two metal dyes (mDOTA-Ho, Ir-DNA-intercalator) and several dozen cells were measured using both modes. We found a ten times higher throughput for single spot analysis, which has as well a straightforward data analysis, shortening the total analysis time further. The signal to noise ratio for single spot analysis was found to be slightly better compared to the signal to noise of pixels in imaging. The mean metal intensity per single cell differed by only 10% between both modes and obtained distributions were found to show no statistically significant differences. Using matrix matched calibration based on standards spotted onto nitrocellulose membrane, we achieved detection limits (10 σ) of 12 fg for Ir and 30 fg for Ho and quantified 57 ± 35 fg Ir and 1,192 ± 707 fg Ho per single cell. Compared to a conventional ICP-MS measurement of a digest of ~60,000 cells, 54 % of Ir content and 358 % Ho content was found using quantitative LA-ICP-MS. The difference might be a consequence of the two metal dyes binding to different structures of the cell and therefore might behave differently in sample preparation for conventional and LA-ICP-MS.