Darkfield illumination is commonly used for the analysis of biological samples containing nanomaterials that significantly scatter light. When combined with hyperspectral imaging, it becomes an exceptional tool to also detect the composition and the location of nanomaterials embedded in cells. IMA™, Photon Etc.’s hyperspectral imager, can be equipped with a highly efficient darkfield condenser and generate high contrast images of biological samples.
The high throughput of Photon Etc.’s hyperspectral filter allows the rapid acquisition of spectrally resolved high-resolution images. Since the camera captures the whole area in the field of view, it is possible to collect spectral and spatial information in real time, with the possibility of recording spectrally resolved videos to follow the dynamics of cells and luminescent nanoscale components. PHySpec™, Photon Etc.'s software, enables principal component analysis (PCA) in order to identify the smallest variations of single and aggregated nanoparticles.
With the purpose of showing the capabilities of IMA™ to analyze nanomaterials in biological systems, a sample of MDA-MB-23 human breast cancer cells has been tagged with 60 nm gold nanoparticles (GNPs) and exposed to a dark field illumination on the entire field of view (Figure 1). With a 60X objective, an area of 150x112 μm2 was imaged, with a step of 2 nm and an exposition time of 2 s per wavelength. The complete analysis took only a few minutes, for more than one million spectra, each of them covering the whole visible spectrum.
Cells typically have a flat scattering spectrum, whereas GNPs show a sharp peak around 550 nm. Figure 2 illustrates the 550 nm image extracted from the dark field hyperspectral cube of the breast cancer. The GNPs are marked with a green colouring after PCA software processing. The magnification of a breast cancer cell (Figure 3a) and the spectra of the regions containing GNPs (some examples in Figure 3b) confirmed the presence of single 60 nm NPs (peak at 550 nm) and their aggregates (peaks red-shifted). The hyperspectral camera did not detect any GNPs in the areas between the cells.
Results kindly provided by David Rioux, Éric Bergeron and Michel Meunier, at École Polytechnique of Montreal, Quebec, Canada.