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Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.
In recent years, elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining in importance. Latest improvements regarding spatial resolution (down to 1 µm) and washout time make LA-ICP-MS particularly interesting for single cell analysis.
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.
LA-ICP-MS was used to study the NP pathway from uptake, via intracellular processing up to cell division. Fibroblast cells were incubated with different metallic NPs under varying experimental conditions. For LA analysis the cells were fixed with formaldehyde and dried.
Our results show that LA-ICP-MS is able to localise NP aggregates within cellular substructures. The 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 [1, 2]. A strong dependence of NP uptake on concentration and incubation time was found. Additionally, the number of NPs internalized by individual cells was determined and variations within the cell population became visible.
A new laser ablation system providing a short washout time (50 ms) together with small spot sizes (< 4 µm) and high repetition rates allows high spatial resolution applications. First results of cell imaging will be shown.
The findings demonstrate the potential of LA-ICP-MS enabling insight into NP uptake and intracellular distribution dependent on experimental parameters.
The traceability and availability of nanoparticles enables their use to enhance a variety of nano-biological and nano-medicinal applications. The particular size and shape of nanoparticles determine the uptake rate and pathway into the cell, and therefore impact specific cell components and processes. Selecting specific particle types allows researchers to target the process or structure of interest, with minimal additional impact. This can be used for drug or DNA delivery, and is being explored for use in oncology. Understanding the different uptake mechanisms and impacted processes requires sub-cellular Imaging resolution to determine, for example, whether or not the nanoparticles are reaching the nucleus.
Sub-cellular imaging has traditionally been challenging to achieve with laser ablation ICP-MS due to a lack of sensitivity at small spots. Bioimaging using LA-ICP-MS is a well-established technique, but usually applied on the tissue scale, which depends on larger spot areas where sensitivity is less problematic. The improved sensitivity and washout from the NWRimage has allowed faster imaging of smaller spots. The NWRimage also provides the possibility of true sub-micron spot sizes. This work compares the capabilities of standard laser Ablation (NWR213 system) with results from the NWRimage platform, which has been optimized for imaging applications.
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.
The interaction of nanoparticles (NPs) with cells has become a major field of interest, ranging from medical applications to nanotoxicology. 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.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is an established quantitative multi-elemental analysis and mapping technique. In recent years, it was shown that LA-ICP-MS can provide quantitative as well as distribution information of NPs in cell samples.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. Additionally, the local distribution of naturally occurring elements in cells like P was measured to indicate cell morphology. The cells were incubated with different types of NPs under varying experimental conditions. For LA analysis, the cells were fixed and dried.
Our findings show, that LA-ICP-MS is suitable for the localisation of 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 physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into NP uptake, intracellular distribution and cell-to-cell variation dependent on experimental parameters.
Nanoparticles (NPs) have found a wide range of applications in research and industry. Thereby the interaction of NPs with biological systems like cells has become a major field of interest, ranging from medical applications to nanotoxicology. Size, shape and surface modification of the nanomaterials determine the uptake rate and pathway into the cells, and therefore impact specific cell components and processes.
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. Improvements concerning both spatial resolution (down to 1 µm) and signal-to-background ratio due to low-dispersion LA chambers make LA-ICP-MS particularly interesting for single cell analysis.
Here LA-ICP-MS was applied for the imaging of individual cells to study the uptake and intracellular processing of metal-containing nanostructures. The cells were incubated with different NPs under varying experimental conditions and afterwards fixed with para-formaldehyde and dried for LA analysis. High-spatial resolution LA-ICP-MS was achieved by careful optimisation of the laser ablation parameters.
Our findings show, that LA-ICP-MS is applicable to localize NP aggregates within cellular compartments. The uptake efficiency depends strongly on the physicochemical properties of the nanostructures as well as on the incubation conditions like concentration and incubation time.
The results demonstrate the potential of LA-ICP-MS providing insight into nanoparticle-cell interaction dependent on experimental parameters.
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.
We applied high resolution laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with cellular spatial resolution for bioimaging of nanoparticles uptaken by fibroblast multicellular spheroids (MCS). This was used to quantitatively investigate interactions of silver nanoparticles (Ag NPs) and the distributions of intrinsic minerals and biologically relevant elements within thin sections of a fibroblast MCS as a three-dimensional in vitro tissue model. We designed matrix-matched calibration standards for this purpose and printed them using a noncontact piezo-driven array spotter with a Ag NP suspension and multielement standards. The limits of detection for Ag, Mg, P, K, Mn, Fe, Co, Cu, and Zn were at the femtogram (fg) level, which is sufficient to investigate intrinsic minerals in thin MCS sections (20 μm thick). After incubation for 48 h, Ag NPs were enriched in the outer rim of the MCS but not detected in the core. The localization of Ag NPs was inhomogeneous in the outer rim, and they were colocalized with a single-cell-like structure visualized by Fe distribution (pixel size of elemental images: 5 × 0.5 μm). The quantitative value for the total mass of Ag NPs in a thin section by the present method agreed with that obtained by ICP-sector field (SF)-MS with a liquid mode after acid digestion.
We investigated the penetration of silver nanoparticles (Ag NPs) into a three-dimensional in vitro tissue analog using NPs with various sizes and surface coatings, and with different incubation times. A high-Resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) time-of-flight (TOF) instrument was applied for imaging the distributions of elements in thin sample sections (20 μm thick). A fibroblast multicellular spheroid (MCS) was selected as the model system and cultured for more than 8 days to produce a natural barrier formed by the extracellular matrix containing collagen. The MCS was then exposed for up to 48 h to one of four types of Ag NPs (∅ 5 nm citrate coated, ∅ 20 nm citrate coated, ∅ 20 nm polyvinylpyrrolidone coated, and ∅ 50 nm citrate coated). Imaging showed that the penetration pathway was strongly related to steric networks formed by collagen fibrils, and Ag NPs with a hydrodynamic diameter of more than 41 nm were completely trapped in an outer rim of the MCSs even after incubation for 48 h. In addition, we examined the impact of these NPs on essential elements (P, Fe, Cu, and Zn) in
areas of Ag NP accumulation. We observed a linear increase at the sub-femtogram level in the total concentration of Cu (fg per pixel) in samples treated with small or large Ag NPs (∅ 5 nm or ∅ 50 nm) for 48 h.
In recent years, elemental imaging of biological samples like tissue thin sections using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining more and more importance. Improvements concerning spatial resolution as well as signal-to-background ratio due to low-dispersion sample chambers make LA-ICP-MS also interesting for single cell analysis.
To evaluate the interaction of nanoparticles (NPs) with cells LA-ICP-MS was applied for the imaging of individual cells. Our findings show, that NP aggregates can be localized within cellular compartments. The uptake efficiency depends strongly on the physico-chemical properties of the nanostructures (size, chemical composition, surface modification), as well as on the incubation conditions (concentration, time).
Moreover, LA-ICP-MS is increasingly becoming an important complementary technique in bioanalysis by using element-tagging strategies to determine biomolecules indirectly. Based on the specific binding between antibodies and their corresponding antigens, proteins and peptides can be detected in tissue or cells using tagged antibodies. As artificial tags metal chelates loaded with lanthanides, polymer-based elemental tags or metal-containing nanoparticles can be used. Thereby LA-ICP-MS is a sensitive detection tool for multiplexed immuno-histochemistry of tissue and cell samples.
Our results demonstrate the potential of LA-ICP-MS to investigate the distribution of naturally occurring elements, administered agents as well as biomolecules by using metal-tagged antibodies.