The overall goal of the following experiment is to provide a nano metrological method for monitoring and quantifying the cellular uptake of carbon nanotubes in a statistically relevant number of cells. This is achieved by first treating the cells with water dispersable, carbon nanotubes, which enter the cells in a concentration dependent manner. These carbon nanotubes are eventually labeled with a fluorescent marker.
Then after fixation, each cell is imaged in the bright field, dark field, and fluorescent channels of a multi-spectral imaging flow.Cytometer. To visualize the distribution of the carbon nanotubes and the level of light absorption, scattered light and fluorescence, the nano tube labeling induces in each cell on a pixel by pixel basis. Ultimately, the intrinsic light absorbance and scattering within the cells allows the localization and quantification of the carbon nanotubes.
So this method can help answer new questions in the nano toxicology and nanomedicine fields, such as the impact and fate of carbon based materials in Liveing cells, and also the pot potential of carbon nanotubes as a drug delivery system Begin by dispersing the carbon nanotubes eventually coupled to a fluorescent label in cell culture water. Then sonicate the nanotubes in a bath water sonicate at 20 degrees Celsius for 20 minutes. Next, dilute the nanotubes at 0, 10, 20, and 50 microgram per milliliter concentrations in complete medium, and then incubate two milliliters of each dilution with the cells of interest in 1 25 square centimeter plate per dilution at 37 degrees Celsius after 20 hours, remove the incubation medium from each plate and rinse the carbon nano tube labeled cells with PBS.
After the wash, trypsin eyes the cells on each plate and then resuspend them in complete DMEM medium. Next, transfer each sample to individual einor 15 milliliter falcon tubes, and then spin down the cells for five minutes at 200 times G and room temperature. Resuspend each pellet in 4%para formaldehyde at four degrees Celsius for one hour.
After fixation, spin the cells down again and resuspend each pelleted sample at 15 times 10 to the fifth cells in 150 microliters of PBS. Then filter the samples through individual 50 micron mesh strainers to acquire images of the cells with an image stream. Multi-spectral imaging flow cytometer.
First, select an adapted magnification and then image samples of interest. Use a 685 nanometer laser to image dark field and set the flow cytometer. Number six channel to collect the dark field emission at a band pass of 745 to 800 nanometers.
Image the samples in the number one brightfield and number six darkfield channels for image analysis. First, create a by parametric dot plot with a width by height aspect ratio versus the cell area. This allows discrimination of the single cells which have a standard area and aspect ratio close to one from the multicellular events, which have a large area and a small aspect ratio, and the debris which exhibit an extremely small area.
After using the cell images to verify that the single cells have been correctly selected, set up a bi parametric dot plot for highly contrasted carbon nano tube labeled cells by plotting the contrast against the root mean square gradient to allow the exclusion of the low root, mean square gradient, low contrast unfocused cells, and the selection of the cells in the focused plane. Next, use the find best feature tool to determine the parameter that provides the best statistical separation between the labeled and non labeled cells. For example, for this representative experiment, the mean pixel object feature is the most suitable.
Then plot this parameter against the normalized frequency to trace a histogram for quantification of the nanotubes taken up by the cells at each experimental concentration. Now plot a bi parametric graph for the mean pixel object on bright field versus the intensity on dark field. To check the correlation between these two parameters, nano tube quantification is also assessed by means of creating on the cell images as the carbon nanotubes appear as intense dark spots In the Brightfield channel, select a restricted range of pixels with a low intensity, for example, between zero and 533 to create the threshold mask, one that fits the dark nanotubes precisely to create a second mask denoted mask two for visualizing the nanotubes that appear bright in the dark field channel.
Select a range of pixels with a high intensity. For example, a 150 to 4, 095 range was chosen for this experiment. Then using the area from either the bright field mask one or the dark field mask two, set up a dot plot to quantify the relative internalization of the nanotubes.
For example, plot the area on channel six corresponding to the dark field with mask two against the area on channel one, corresponding to the bright field with mask one. After determining the linear correlation between mask one and mask two, visualize the nanotubes within the cytoplasm by first creating a mask of the entire cell and then erode the mask. Now denoted erode M 0 1 7 by seven pixels to view the interior of the cell.
To visualize the carbon nanotubes within the cell membrane, use the Boolean equation M zero one and not erode M 0 1 7. That is the mask of the entire cell, subtracted from the interior of the cell to create a mask of the membrane only to evaluate the dark pixels on the membrane. Apply the brightfield mask one to the membrane mask.
Apply all of these masks to multiple cells at a time to confirm a distinction between the labeled and unlabeled samples. Then select the black pixels on the membrane with the feature area tool to quantify the carbon nanotubes on the cell membrane. Finally, to discriminate the carbon nanotubes that have been internalized from those that have absorbed onto the membrane.
Plot, the black area of the membrane against the black area of the entire cell. Once all plots have been created for one experimental condition, create a statistical report template. Save the template as an A ST file and batch all the data files.
The image stream device produces multiple high resolution images of each cell in the flow cytometer, including brightfield, darkfield, and fluorescence channels, as demonstrated here with three different nano tube labeled cells. The overlays of these channels are also shown. The presence of carbon nanotubes induces changes in both the contrast and the gradient of labeled cells compared to unlabeled cells.
Thus, the creation of a bi parametric dot plot is important for selecting the focus cells. Unfocused cells in yellow can be discriminated by their low contrast and low root mean square gradient regardless of the presence or absence of nano tube labeling. Unlabeled focus cells also exhibit a low contrast, but demonstrate a high root, mean square gradient when the focus cells are labeled with nanotubes, however, the cells demonstrate a high contrast, low root, mean squared gradient profile.
Internalized carbon nanotubes are easily distinguished, particularly in the Brightfield channel, where they appear as intense dark spots, the areas of which increase with the concentration of nanotubes. When the mean pixel signals of the brightfield images are analyzed, a relative quantification of the nano tube uptake can be determined. For example, these histograms demonstrate the significant increase in nano tube uptake that occurs with each increase in the nano tube labeling concentration as demonstrated carbon nano tube quantification is also possible through the use of masks applied either on a restricted range of dark pixels within a bright field image, or within the high intensity pixels within the dark field, corresponding to the areas where the nanotubes are present.
Plotting the area of these masks then allows further quantification of carbon nano tube uptake. Additionally, the correlation between the spots in the bright and dark field images can be evaluated by plotting the dark field intensity versus the bright field mean pixel signal, or by plotting the area of the mask within the bright field versus the area within the dark field. In these next two figures, analyses of the carbon nanotubes localized within the interior of the cell are shown.
These first images demonstrate the different masks that were created. Note that the robustness of the localization masks based on the erosion of the default cell mask must be checked visually on the collection of the labeled and unlabeled cells. As just demonstrated in these dot plots, the total area of the black spots on the cell membrane versus that of the entire cell have been plotted to determine the internalization score.
For cells that have been incubated for 20 hours at 37 degrees Celsius compared to cells that were incubated for two hours at four degrees Celsius at four degrees Celsius, the carbon nanotubes are found mostly located on the membrane, whereas at 37 degrees Celsius, the carbon nanotubes are internalized in more than 90%of the cells. Carbon nano tube fluorescence is not a good indicator of carbon nano tube internalization due to a too low correlation between the fluorescent spots and the spots within the bright and dark field channels. Indeed, the correlation between the fitzy intensity and the bright field mean pixel indicates a Pearson correlation coefficient of minus 0.2.
As demonstrated in these images, the fluorescent signal does not reliably match up with the bright field black spots, carbon nanotubes, confinement, and aggregation within intracellular lysosomes partially quenched the fluorescent signal. In contrast, dispersed nanotubes may give a more reliable fluorescent signal After its development. This technique paves the way for researchers in the field of nanotechnology and nanomedicine to explore the interaction of living cells with carbon based nanomaterials or any nanomaterials with a high absorption and life scattering ability using a statistical high throughput cytometry method.
