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The analytical potential of a nanosecond laser ablation inductively coupled plasma mass spectrometer (ns-LA-ICP-SFMS) system, equipped with an ultra-fast wash-out ablation chamber, is critically investigated for fast and highly spatially resolved (∼μm) qualitative elemental distribution within single cells. Initially, a low surface roughness (< 10 nm) thin In–SnO2 layer (total coating thickness ∼200 nm) deposited on glass is employed to investigate the size, morphology and overlapping of laser-induced craters obtained at different laser repetition rates, making use of Atomic Force Microscopy (AFM). Conical craters with a surface diameter of about 2 µm and depths of about 100 nm were measured after a single laser shot. Furthermore, the influence of the sampling distance (i.e. distance between the sample surface and the inner sniffer of the ablation chamber) on the LA-ICP-MS ion signal wash-out time is evaluated. A significant decrease of the transient 120Sn+ ion signal is noticed after slight variations (±200 μm) around the optimum sampling position. Ultra-fast wash-outs (< 10 ms) are achieved reducing the aerosol mixing from consecutive laser shots even when operating the laser at high repetition rates (25 – 100 Hz). Fast and highly spatially resolved images of elemental distribution within mouse embryonic fibroblast cells (NIH/3T3 fibroblast cells) and human cervical carcinoma cells (HeLa cells), incubated with gold nanoparticles (Au NPs) and Cd-based quantum dots (QDs), respectively, are determined at the optimized operating conditions. Elemental distribution of Au and Cd in single cells is achieved using a high scanning speed (50 µm/s) and high repetition rate (100 Hz). The results obtained for the distribution of fluorescent Cd-based QDs within the HeLa cells are in good agreement with those obtained by confocal microscopy. The size, morphology and overlapping of laser-induced craters in the fixed cells are also investigated using AFM, observing conical craters with a surface diameter of about 2.5 µm and depths of about 800 nm after a single laser shot.
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.
This work demonstrates the sub-cellular resolution bio-imaging capabilities of the NWRimage system with Dual Concentric Injector (DCI) technology in a practical application. 3T3 fibroblast cells incubated with gold nanoparticles were imaged, demonstrating the sub-cellular imaging capabilities. A laser ablation system specifically designed for rapid, high resolution imaging was employed. The system´s optical layout is optimized to minimize crater diameters. Sensitivity and speed were achieved using the DCI technology for ultra-fast washout times.
We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg/mL for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure.
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.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is nowadays a versatile and powerful analytical method for direct solid sample analysis. The applicability has been demonstrated for a wide variety of samples covering hard and soft materials. In an imaging mode the technique provides quantitative information on the elemental distribution within a sample. LA-ICP-MS imaging is of particular interest in biomedical research as the distribution of an element gives valuable insight on uptake and distribution of essential and toxic trace elements, administered contrast agents as well es nanoparticles. LA-ICP-MS is therefore a powerful complement to other imaging techniques. Recent instrumental improvements, especially in sample chamber design, have contributed to better sensitivity and spatial resolution enabling subcellular imaging. The book chapter provides a comprehensive overview about spatially resolved localisation and quantification of various nanoparticles in cells and tissue thin sections by LA-ICP-MS. Furthermore, different sample preparation strategies and internal standardisation and calibration approaches for bioimaging by LA-ICP-MS are summarized and discussed.
Metal-containing nanomaterials are used in numerous fields ranging from industrial applications to nanomedicine. Several studies have demonstrated that the physicochemical properties of nanoparticles have an impact on their pharmacokinetics, transfer and clearance. The high sensitivity and multielement capability of LA-ICP-MS enables the elucidation of interactions between tissue components and nanomaterials used as imaging probes or drug carriers. Potential toxic effects are investigated as well. Thus, LA imaging significantly supports the clinical translation of safe and efficient nanoparticles for diagnostic and therapeutic purposes.
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.
Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
Prostate cancer (PCa) is one of the most common cancers in men. For detection and diagnosis of PCa, non-invasive methods, including magnetic resonance imaging (MRI), can reduce the risk potential of surgical intervention. To explore the molecular characteristics of the tumor, we investigated the applicability of ferumoxytol in PCa in a xenograft mouse model in two different tumor volumes, 500 mm3 and 1000 mm3. Macrophages play a key role in tumor progression, and they are able to internalize iron-oxide particles, such as ferumoxytol. When evaluating T2*-weighted sequences on MRI, a significant decrease of signal intensity between pre- and post-contrast images for each tumor volume (n = 14; p < 0.001) was measured. We, furthermore, observed a higher signal loss for a tumor volume of 500 mm3 than for 1000 mm3. These findings were confirmed by histological examinations and laser ablation inductively coupled plasma-mass spectrometry. The 500 mm3 tumors had 1.5% iron content (n = 14; sigma = 1.1), while the 1000 mm3 tumors contained only 0.4% iron (n = 14; sigma = 0.2). In vivo MRI data demonstrated a correlation with the ex vivo data (R2 = 0.75). The results of elemental analysis by inductively coupled plasma-mass spectrometry correlated strongly with the MRI data (R2 = 0.83) (n = 4). Due to its long retention time in the blood, biodegradability, and low toxicity to patients, ferumoxytol has great potential as a contrast agent for visualization PCa.
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.