In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.
Nanomateriales a base de carbono, como los nanotubos de carbono (CNT), pertenecen a este tipo de nanopartículas que son muy difíciles de diferenciar de las estructuras celulares ricos en carbono y de hecho todavía no hay método cuantitativo para evaluar su distribución en los niveles de células y tejidos. Lo que proponemos aquí es un método innovador que permite la detección y cuantificación de los nanotubos de carbono en las células usando un flujo de imágenes multiespectrales citómetro (ImageStream, Amnis). Este dispositivo recién desarrollado integra tanto un alto rendimiento de las células y formación de imágenes de alta resolución, proporcionando de este modo imágenes para cada celda directamente en el flujo y por lo tanto el análisis de imágenes estadísticamente relevante. Cada imagen de la célula se adquiere en campo claro (BF), campo oscuro (DF), y los canales de fluorescentes, que da acceso, respectivamente, con el nivel y la distribución de la absorción de la luz, la luz dispersa y fluorescencia de cada célula. El análisis consiste entonces en una comparación píxel por píxel de cada imagen, de la 7,000-10,000 células adquiridas para cada condición del experimento. La localización y cuantificación de los nanotubos de carbono se hace posible gracias a algunas propiedades intrínsecas particulares de CNT: fuerte absorción de luz y la dispersión, de hecho CNT aparecen como absorbe fuertemente las manchas oscuras en BF y puntos brillantes en DF con una colocalización precisa.
Esta metodología podría tener un impacto considerable en los estudios acerca de las interacciones entre los nanomateriales y las células ya que este protocolo es aplicable a una amplia gama de los nanomateriales, en la medida en que son capaces de absorber (y / o difusión) con suficiente fuerza a la luz.
During the last decades, the place of nanoparticles has gained in importance in a wide range of applications. In particular, carbon nanotubes (CNTs) are more and more explored as nanotools for diverse diagnostic and therapeutic applications; thanks to their particular electrical, thermal, and spectroscopic properties, CNTs can act as delivery vehicles for varied drugs or contrast agents, or can also be involved in tissue engineering1.
However, concerns about the potential toxicity of such nanomaterials are still under debate, mainly because the fate of CNTs in the organism remains very controversial. There is indeed a lack of appropriate methods to detect and quantify them in live cells and tissues: for example, techniques such as Raman spectroscopy, photoacoustic, and near-infrared photoluminescence imaging have been suggested, but each method shows limitations depending on the type of nanotubes and/or their environment2-8.
Here, the method proposed could be applicable for the detection and quantification of nanoparticles in cells. This was made possible by the use of ImageStream, a device combining both flow cytometry and high resolution imaging. It couples the advantages of acquiring a large among of cells (up to 5000 events/sec) – and benefits of statistical data and high resolution images of each cell at the same time (0.5 µm spatial resolution). Generally, images are collected using a CCD camera and are a two dimensional grey scale representation of the cell. The light is quantified for each pixel in the image and identifies both the intensity and the location of either fluorescence or components with a subcellular size (in BF for example), allowing thus a powerful ability to discriminate cells based on their appearance9,10.
The aim was here to design a nanometrology method which would enable a quantitative assessment of the distribution and behavior of CNTs in cells. In particular, this method was developed to study cellular uptake, processing and exocytosis of CNTs by cells. We evidenced first the complex cellular trafficking of CNTs and secondly the transfer of these nanomaterials between cells: cell-internalized CNTs escape their host cells when stressed, spread abroad in the extracellular space and are reinternalized by naïve cells, whether they are homotypic or heterotypic11
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Previous study showed that different mechanisms of cell uptake, either active or passive ones, could occur depending on CNT functionalization and aggregation state12. Here the multi-walled CNTs were functionalized through 1,3-dipolar cycloaddition and amidation and then derivatized with a fluorescent probe, fluoresce in isothiocyanate (FITC). On transmission electron microscopy, they appear to be internalized by cells either individually with cytosolic localization or as bundles, mostly found in endosomal and lysosomal compartments11. On ImageStream, the clustered CNTs appear with a high correlation as dark spots on BF and bright spots on DF, allowing a clear distinction between CNT-labeled cells and control cells. On the other hand, the distribution of the fluorescent signal emitted by the fluorescent marker functionalized with CNTs was not as well colocalized: indeed, likely due to a quenching phenomenon, the green fluorescent spots do not necessarily match the spots on BF and DF. This indicates that, firstly, a fluorescent probe is not mandatory for the detection of CNTs with the ImageStream, and secondly – a fortiori – that it is not fully representative for both quantification and localization of CNTs.
Beyond giving a statistical relative quantification of nanoparticles, this method also provides information concerning the localization of CNTs in cells, meaning that it discriminates, for instance, CNTs on the interior of the cell versus those which are bound to the plasma membrane. The ability to localize CNTs on cell images clearly overcomes the limitation of conventional flow cytometry analysis13. The procedure was developed here via the creation of masks fitting with specific regions of the cell and selecting also the dark spots corresponding to loaded CNTs.
Generally speaking, this method could be applied for a large range of experiments involving the need of visualization, or quantification, in cells on condition that the nanoparticles used are able to strongly absorb (and/or scatter) the light.
1. Modification and Troubleshooting
In this study, the first objective was to evaluate the quantity and the localization of CNTs in cells. The confocal microscopy could have been a priori the gold method to assess the intracellular distribution of CNTs functionalized with the fluorescent marker FITC8,14. However, the main drawback is the limitation to single cell imaging and, therefore, a lack of statistically relevant data. The ImageStream allows work beyond these obstacles: indeed, by screening a large number of cells (5,000-10,000) in a short time (<min), it yields statistically robust results, enhanced also by the high resolution imaging which provides images for each cell with a subcellular resolution (0.5 µm). It makes then possible to analyze a large amount of data based on single image analysis.
In comparison to conventional flow cytometry, the unique advantage of ImageStream is to provide multispectral images of each cell and a pixel by pixel comparison of signal intensity. Thus ImageStream combines the statistical power of high throughput flow cytometry with the analytical advantages of confocal imaging for each analyzed cell. It is crucial for a label-free relative quantification and localization of CNTs in cells.
2. Critical Steps Within the Protocol
The critical step consists of the choice of parameters most suitable of each study. In our case, the best way was to base the analysis on the absorption and scattering properties of the nanotubes: the good correlation between the localization of dark spots on BF and bright spots on DF supports that the developed method is relevant and reliable – and may not have been possible without cell imaging.
3. Limitations of the Technique
It must be emphasized that the visualization of CNTs in cells was possible only because they tend to accumulate in endosomal intracellular vesicles, and then appear as strong absorptive and scattering spots. Individual nanotubes should not be detected based on this method whereas it could be eventually detected on fluorescence, which constitutes a clear limitation. Second, the quantification is not 'absolute' but relative for different labeling concentrations and in comparison to the control unlabeled cells. Some quantitative features (mean pixel, intensity, etc.) are absolute parameters, whereas others depend on the choice of the masks (threshold mask, localization masks).
4. Significance with Respect to Existing Methods
In regards to fluorescence, the ImageStream analysis highlights the poor colocalization of CNT clusters between FITC bright signal and spots on the others channels. Therefore, it appears that fluorescence was not a reliable indicator for the quantification and localization of CNTs. Nevertheless, overcoming the limitation of conventional flow cytometry, which is mainly based on fluorescence quantification, the multispectral high throughput ImageStream paves the way for a statistical label-free quantification, while providing localization of carbon nanotubes inside the cells.
5. Future Applications
This high throughput statistical nanometrology method could be generalized to any nanomaterials, which show high absorption and scattering of light when interacting with cells. Therefore, it may have important applications for investigations of the nano-bio-interface, nanotoxicology, and nanomedicine.
The authors have nothing to disclose.
This work was supported by ANR P2N (Nanother project 2010-NANO-008-04), by the Region Ile de France (contract no E539), and by CNRS. Open Access was paid for by EMD Millipore.