Nanoparticles are increasingly used in industry and medicine due to their unique physiochemical properties. However, concerns for human health associated with prolonged nanoparticle exposure persists. These risks can be quantified by studying the behavior of nanoparticles inside cells and the induced metabolic responses of cells to nanoparticles.

This is partially due to the lack of methods that allow the detection and quantification of internalized nanoparticles in a sincle cell. While numerous analytical tools, including microscopy and mass spectrometry, can be used to estimate the cellular uptake of nanoparticles, these only provide qualitative information at a microscopic level. On the contrary, in situ methods based on atomic spectroscopy can reduce the quantity of imaging artifacts that arise from sample preparation and can yield direct observation of nanoparticles.

To take advantage of this, we present a correlative approach which allows us to study nanoparticles either in the native state or lab bed with a fluorescent tag. Here, we describe a method based on the combination of fluorescence microscopy and nuclear microprobe analysis. This provides images of the cellular density, chemical element special distribution, and the amount of nanoparticles per cell.

As a demonstration, we shall research from cells exposed to titanium dioxide nanoparticles over 24 hours using both fluorescence microscopy and nuclear microprobe analysis. For this protocol, a custom sample holder made with the polymer PEEK is needed. It must be suited for cell culturing and in vitro observations.

Cover the holder with two micron thick polycarbonate foil. The polycarbonate foil is glued to the PEEK dish with a thin layer of Formvar solution. Once mounted, the sample holder needs to be sterilized.

Multiple sterile sample holders may be stored, one per well, in a sterile twelve-well plate until they are needed for use. Cells transfected with matrix row GFP plasmids are checked by fluorescence microscopy to insure transfection has occurred successfully, and that they express the fluorescent protein. Harvest the cells with trypsin, and incubate them for three minutes at 37 degrees Celsius.

Stop the trypsin action by adding fresh culture medium. Pellet the cells by centrifugation for five minutes at 1200 revolutions per minute at four degrees Celsius. Remove the supernatant and add an appropriate volume of fresh culture medium.

Count the cells and perform a dilution with fresh and thermostated complete culture medium to obtain a cell suspension of 500 cells per microliter. Plate a 40-microliter drop at the center of the polycarbonate foil. Carefully place the sample in the cell incubator for two hours.

Gently add two milliliters of fresh culture medium and leave them for 24 hours. Fluorescent dimodified titanium oxide nanoparticles were designed, synthesized, and grafted with commonly used fluorophores in order to detect, track, and localize nanoparticles in vitro. For this step, a titanium dioxide nanoparticle suspension in ultra-pure water with a concentration of one milligram per milliliter should already have been prepared.

Disperse the nanoparticles by using one-minute long intense sonication pulses at room temperature. Dilute the nanoparticles in an appropriate culture medium to obtain an exposure suspension of four micrograms per square centimeter. Switch the previous cell culture medium to this new nanoparticle-containing medium and mix it gently to achieve a homogeneous nanoparticle distribution.

Prepare a set of control cells in the same manner without the addition of titanium dioxide nanoparticles. Incubate the cell populations for 24 hours. Using paraformaldehyde, these samples may be fixed and processed for in situ and single-cell imaging using fluorescence microscopy.

Cryogenic physical fixation, however, preserves the cell ultrastructure and biochemical integrity. Plunge freezing fixation rapidly stops the cellular activity within milliseconds and avoids using fixative compounds. To cryogenically fix the cells, we perform the following.

Prepare an aluminum transfer plate by cooling it in liquid nitrogen. Store the plate in a box filled with liquid nitrogen, keeping the plate surface above the liquid in the cold nitrogen vapor. Chill a sufficient amount of 2-methylbutane to 150 degrees Celsius.

Prepare one 50 milliliter Falcon with culture medium and two Falcons with ultra-pure water. Ensure absorbent paper is nearby to dry the sample before freezing it. Rinse cells once in culture medium and then two more times in sterile, ultra-pure water to remove excess salts remaining in the culture medium.

Quickly dry the sample on the absorbent paper. Plunge-freeze the cells in the chilled 2-methylbutane for 30 seconds and place them on the chilled aluminum transfer plate. It is critical that the box be kept closed as much as possible in order to prevent water vapor from condensing on the cold plate surface.

After freezing all samples, freeze-drying is performed by the following method to sublimate away any water. First, perform a primary desiccation for 12 to 24 hours at low pressure and low temperature. Next, keeping the pressure low, perform a secondary desiccation phase for at least 24 hours with the plate temperature increased to 40 degrees Celsius.

After cryofixation, samples can be stored at room temperature for several days in sterile and dry conditions protected from dust and moisture. Nuclear microprobe analysis was conducted at the Ifera facility in Bordeaux, France. A 3.5 megavolt singletron particle accelerator delivers a light ion beam in the megaelectron volt energy range.

Chemical element imaging was carried out on the microprobe beamline using complementary ion beam analytical techniques. The techniques used through a micro-PIXE, particle-induced X-ray emission, STIM, scanning transmission ion microscopy, and micro-RBS, rutherford backscattering spectrometry. Samples are positioned inside the analysis chamber and irradiated under vacuum with different particles in order to perform different ion beam analyses.

STIM is used to record aerial density maps of cells based on the energy particles loose passing through regions of cells with different densities. Micro-PIXE and micro-RBS analysis provide the spatial distribution and quantification of chemical elements at a single-cell level. A 1.5 megaelectron volt proton microbeam is focused down to a diameter of about 1 micrometer and it is scanned across the cells of interest, identified by STIM.

X-rays emitted from atoms present in the sample, ranging from sodium to titanium, permit the elemental concentrations to be determined. Backscattered protons are collected in order to measure the total number of incoming particles required to normalize X-ray intensities. It is critical to have certified calibration standards for the quantification of elemental concentrations in order to calibrate the X-ray detector response.

In order to the analyze the ion beam data, we developed a new plugin for ImageJ. First, STIM density maps were calculated. Contrast in scanning transmission iron microscopy images is due to local differences in density and allows the detection of cell structures such as the nucleus and cytoplasm.

Several maps can be processed at once. Each map corresponds to the medium transmitted energy from incoming particles. The second step in data analysis consists in calculating individual cell spectra.

Particle-induced X-ray emission analysis yields both the chemical composition of the sample and the elemental maps of chemical elements. Chemical element maps are computed after sorting photons according to the beam position at the time of recording and selecting an energy window centered around a specific element. Maps usually represent the number of detected events at the beam position and contain quantitative data.

A stack of chemical maps, one for each selected element, is calculated. Regions of interest for each cell are found using the PIXE data for phosphorus. The spectra corresponding to each whole cell is then calculated by summing the individual spectra across each cell's region of interest.

From here, quantitative data about chemical abundances in each cell can be extracted using dedicated PIXE and IBS analysis software. Here we show fluorescence imagery of cells after paraformaldehyde fixation. The top row shows cells that have not been exposed to titanium dioxide nanoparticles while the bottom row has been.

The blue channel shows the cell nucleus, the green channel shows mitochondria, and the red channel shows titanium. It can be clearly seen that no titanium dioxide is present in the controls. From the merged channel at the right, it can be seen that nanoparticles are found exclusively in the cytoplasm and are excluded from mitochondria.

What this method lacks, however, is quantitative information about the nanoparticle concentration. Nuclear microprobe analysis can hope to provide this. These images have all been taken using various microprobe analysis techniques.

Again, the top row shows the control cells and the bottom row shows cells containing titanium nanoparticles. The leftmost image shows STIM measurements of the density of the cryogenically fixed cells. The following three panels show the abundance of potassium, phosphorus, and titanium present in the cells obtained by PIXE.

Again, no titanium is visant in the controls nor in the cell nuclei. In order to quantify, cell by cell, the uptake of titanium, we define regions of interest based on the cell boundaries shown in the rightmost panel and measure the elemental abundances in these regions. From these PIXE measurements, we have plotted the distribution of elemental abundances at the single-cell level for the control and the exposed populations.

The control population is plotted in white, and the exposed population is plotted in yellow. The median content of titanium present here is quite low compared to the four microgram per square centimeter exposure dose. The range of titanium uptake shows a large variation across the population, from 0.2 micrograms per square centimeter up to 1.8 micrograms per square centimeter.

We also noticed an increase of free intracellular ions such as potassium and calcium in nanoparticle-exposed samples, suggesting an alteration to cellular homeostasis induced by the presence of titanium dioxide nanoparticles. Nuclear microprobe analysis can provide useful information on the subcellular level and provides a quantification of the chemical elements that make up a biological sample. These techniques present many advantages.

One, in sample preparation which does not require chemical fixation. Two, the possibility to study larger areas with the ability to focus on additional regions of interest. Three, the quantification of chemical elements with a sensitivity of a few micrograms per gram.

And four, the identification of cell compartments such as the nucleus, nucleolus, and cytoplasm. The accurate determination of those, when studying the internalization of nanoparticles in single cells, is essential for quantitative nanoparticle toxicology. As shown in the cases studied here, the ability to observe and quantify nanoparticles within individual cells allow us to better understand the value accumulation of endogenous and exogenous chemical elements, such metal oxide nanoparticles.

This protocol highlights the suitability of nuclear microprobe analysis for future studies of nanoparticle interaction with living cells. The quantitative approach gives information about the impact of these nanoparticles in terms of detection, identification, localization, and quantification, and in single-cell level for both native and chemical modified-nanoparticles.