Hyperspectral Imaging as a Tool to Detect and Characterize Nanoparticles in Complex Biofluids

Marina Mulenos George, Christie Sayes, Bernd Zechmann
2020 Microscopy and Microanalysis  
With recent technological advances in hyperspectral imaging, a corresponding interest in cellular uptake of nanoparticles has increased. Previously in the literature, transmission electron microscopy (TEM) was used to monitor where nanoparticles accumulate in the cell. However, this technique is labor intensive and not cost efficient for the general lab. Hyperspectral imaging is user friendly, cost effective, and the sample preparation is less labor intensive compared to TEM. Many studies in
more » ... Many studies in the literature show that nanoparticles of different composition, charge, or morphologies can accumulate in different cellular compartments, but these studies have been limited to engineered particles. There is an increasing need to investigate where biologically transformed nanomaterials will accumulate inside tissues and cells. Here, we studied biotransformed silver nanoparticles (AgNPs) of either positive, negative, or neutral charge in the 50 nm size range. Each particle system was incubated in blood serum for 2 C and then characterized with transmission electron microscopy for morphological assessment and dynamic light scattering to determine particle size. Transmission electron microscopy methods included depositing a formvar coated copper grid on 10 μL f pended AgNP f five min e . T e g id w ll wed d y and then was imaged on a JEOL JEM-1010 transmission electron microscope at 60kV. Human hepatoma cells, HepG2, were grown in a cell treated four-well chamber slide and exposed to the tr n f med n n p i le f C with 5% CO 2. For fluorescent imaging preparation, the cells exposed to transformed AgNPs went through fixation, permeabilization, blocking, staining with three dyes (MitoTracker Red CMXRos to stain mitochondria red, NucBlue Live ReadyProbes to stain the DNA rich nucleus blue, and ActinGreen 488 ReadyProbes to stain F-actin green), and then set with antifade mountant using the Image-iT Fixation/Permeabilization kit from ThermoFischer Scientific. (Fig. 2A) . For hyperspectral imaging analyses, a CytoViva Hyperspectral Microscope was used in fluorescent mode to determine a region of interest and then in enhanced dark field mode to complete hyperspectral imaging. Hyperspectral scans had an exposure time of 0.25s in full mode (600 scans per acquisition area.) Spectra were recorded of nanoparticles in the selected regions of interest and analyzed. Our results indicate that the transformed AgNPs which released ions had an increase in cell penetration. The surface charge directly influenced the uptake mechanism in which the transformed particles entered the cell. These findings are consistent with the current literature in both in vitro and in vivo studies. (1-3) The spectral maps were significantly different from each other due to charge variation. Furthermore, the concentration of silver uptake into the cell was quantified via ICP-MS methods where the neutral particles were seen to have the largest concentration of particle or produced particle ions in the cell. The information obtained from these studies provides crucial insight into colloidal stability of nanoparticles, provides read-across comparisons between engineered and transformed metal-based particles, and aids
doi:10.1017/s1431927620022667 fatcat:wnvl4oxrk5e5ti5asfi4ta7mkq