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Many current nanomaterials can serve as contrast agents in cellular or tissue imaging, drug delivery vehicles or therapeutics, whereas others can cause toxic effects. In order to evaluate nano-bio interactions, the number of nanoparticles (NPs) inside cells as well as their localisation within cellular substructures is of particular interest. The cellular uptake depends on the primary characteristics of the NPs (e.g. size, shape, surface coating) and on the cell type.
Laser ablation inductively coupled plasma mass spectrometry (LA‑ICP‑MS) is more and more used to study the NP pathway from uptake, via intracellular processing up to cell division. High-spatial resolution laser ablation at single cell level is achieved using novel low-dispersion LA chambers and by careful optimisation of laser energy, ablation frequency and scan speed at small laser spot sizes down to 1 µm. Different examples from BAM, Division 1.1 and cooperation partners using LA-ICP-MS to localize and quantify metal-containing nanoparticles are shown. The results demonstrate the potential of LA-ICP-MS providing insight into NP uptake, intracellular distribution and cell-to-cell variation.
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 (10s) of 12 fg for Ir and 30 fg for Ho and quantified 57 +/-35 fg Ir and 1192 +/- 707 fg Ho per single cell.
Compared to a conventional ICP-MS measurement of a digest of about 60000 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.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.
Nanoparticles (NPs) have potential applications in medical diagnostics, imaging, drug delivery and other kinds of therapy. Furthermore, studies concerning nanoparticle uptake by cells are important for risk assessment. Size, shape and surface modification of the NPs determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes. Understanding the different uptake mechanisms and involved processes require sub-cellular resolution to determine, for example, whether the nanoparticles are reaching the nucleus. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. However, sub-cellular imaging has traditionally been challenging to achieve due to a lack of sensitivity at small laser spots. But now novel laser ablation systems with improved sensitivity and washout time allow imaging at high lateral resolution with spot sizes down to 1 µm.
Here LA-ICP-MS was applied for the imaging of individual fibroblast cells to study the uptake and intracellular processing of metal-containing NPs. To indicate cell morphology the local distribution of naturally occurring elements in cells like P and Zn was measured, too. Our results show that LA-ICP-MS can be used to localise nanoparticle aggregates within cellular compartments. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, but do not enter the cell nucleus. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time. The potential of LA-ICP-MS for analysis at single cell level will be demonstrated.
Studying the interaction of nanoparticles (NPs) with cells has become a growing field of interest. Research topics are ranging from nanotoxicology to medical applications e.g. as theranostic agents. In order to evaluate nano-bio interactions, the number of NPs inside cells as well as their localisation within cellular substructures is of particular interest.
In recent years, elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining more and more importance. Latest instrumental developments regarding spatial resolution (down to 1 µm) and detection efficiency make LA ICP-MS particularly interesting for single cell analysis.
Here, we have applied LA-ICP-MS for sub-cellular scale imaging of individual cells to study the NP pathway from uptake, via intracellular processing up to cell division. Furthermore, the local distribution of naturally occurring elements in cells like P was measured to indicate the cell morphology.
Murine fibroblast cells were incubated with different metal-containing NPs under varying experimental conditions. For LA analysis, the cells were fixed and dried. Sub-cellular resolution was achieved by careful optimisation of the laser ablation parameters. By rastering with the laser beam across the sample, a two-dimensional image of the elemental distribution can be received. Our results show that LA-ICP-MS is able to localise NP aggregates within cellular substructures. The studied NPs accumulate in the perinuclear region in the course of intracellular processing, e.g. multivesicular fusion and endosomal maturation, but do not enter the nucleus. The uptake depends on the physico-chemical properties of the nanostructures and on the incubation conditions like concentration and incubation time. Additionally, the number of NPs internalized by individual cells was determined and variations within a cell population became visible.
The findings demonstrate the potential of LA-ICP-MS providing insight into NP uptake and intracellular distribution dependent on experimental parameters.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is nowadays an established multi-elemental analysis and mapping technique. It was shown that LA-ICP-MS can visualize the elemental distribution within tissue thin sections or cell samples. Quantification is possible by using appropriate matrix-matched calibration samples. Besides naturally occurring elements and metals from contrast agents, biomolecules using metal-tagged antibodies were detected in different bio-medical samples. By combining the results with findings from histology, magnetic resonance imaging (MRI) and other techniques disease related changes like alterations of the extracellular matrix can be investigated.