Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

The overall goal of this procedure is to identify metal oxide nanoparticles in histological samples by dark field microscopy and hyperspectral mapping. This is accomplished by first identifying a region of interest in a positive control sample containing nanoparticles and capturing a dark field image. The second step is to create a hyperspectral image or data cube of the region of interest using the hyperspectral camera.

Next, a reference spectral library is created from the positive control data cube, which is filtered against a negative control to remove any false positives. The final step is to use the reference spectral library to map against experimental samples to identify and locate nanoparticles of the same composition. Ultimately enhanced dark field microscopy and hyperspectral mapping are used to identify metal oxide nanoparticles in histological samples in a more rapid and less resource intensive manner than other techniques such as electron microscopy.

This method offers several advantages over conventional nanomaterial imaging and characterization techniques such as electron microscopy. In that sample preparation is typically minimal and non-destructive. Image acquisition and analysis is less time and cost intensive, and it can be applied for nanoparticle imaging in a variety of matrices.

Individuals knew the methodol sometimes struggle because creating the appropriate positive control samples and reference spectral libraries is both challenging and time consuming. But once those initial steps are completed, experimental samples can be assessed pretty quickly. While this method can provide insight into cutaneous penetration of nanoparticles, it can also be applied to other biological samples such as bronchoalveolar lavage and blood smears, as well as non-biological samples, such as filters used in occupational exposure assessments.

Demonstrating this technique will be Plar Sosa, a medical student from my laboratory Begin by Plugging in and turning on the light source, the stage controller, the optical camera, the hyperspectral camera, and the computer system. Then raise the stage and condenser to the operating position and carefully apply three to five drops of Type A immersion oil onto the condenser lens. If any bubbles are created, wipe away the oil and reapply it to the lens.

Next, position the sample on the stage and slowly raise the condenser until the immersion oil makes contact with the underside for the slide. This will be noticeable through the rapidly brightening ring of illumination where the oil makes contact with the slide. With the condenser in place, switch to the 10 x objective and then focus and align the condenser to maximize the brightness if needed, adjust the condenser alignment knobs to center the bright spot.

Open the optical imaging software. Focus the sample using the fine objective focus knob. Then find a region of interest.

Adjust the condenser focus as necessary to equilibrate the illumination. Click on the settings tab in the menu bar and click on image capture button. For capture event.

Select the desired image format and assign a file name for the image. Additionally, select the default time lapse value, and finally click okay. Next, go to the exposure menu and select the exposure settings that create the highest contrast image.

Here, the level is set to 0.0%The gain is set to 2.8 decibels and the shutter speed is set to 56 milliseconds seconds. Capture the image by clicking the image button in the menu bar. Change the objective to 40 x and 100 x.

Capturing images at higher magnifications, ensure the light guide is directed to the Hyperspectral camera, and then open the Hyperspectral imaging software for acquisition of the HSI data cubes in the software open hyperspectral microscope in the menu bar and select HSI microscope controls. Here set the objective magnification and the safe path. Next, change the area capture in settings, and change the number of lines.

In this study we used 720 set the exposure time. Here we used 0.25 seconds. Leave everything else to default and click on preview HSI to view the image.

Observe the resulting intensity graph representing the intensity of the highest wavelength as the user moves across the x axis of the center of the camera's field. Move the cursor to generate an accurate outline of the graph. This will cause a numerical intensity indicator to appear on the left.

Focus the image based on this preview by adjusting the fine objective focus to sharpen the peaks in this image. Next, optimize the light intensity by adjusting source brightness condenser focus, or cancel the preview and adjust the exposure time in the software. Click capture and observe as four new windows with the names available.

Bands list. Number one zoom. Number one, scroll and number one RGB band appear.

Maximize the number one RGB band window as this is the data cube, which will be used for all future references.Right? Click this data cube, save it as a tiff file and click okay. Right click in the data cube image window and left Click on Z profile spectrum.

When the pop-up spectral profile window appears left, click on the pixels of interest on the data cube target. The brightest ones are those that can be confidently identified as representing the material of interest. Under the particle analysis menu, select the particle filter tool to identify particles present in the data cube.

In the new popup window set, the spectral max must exceed value, so it is higher than background pixels, but lower than the materials of interest. The valid data max is 16, 000. In the particle filter review tool window, click select all.

Then click export to spectral library and assign the name for your spectral library. After selecting a sample to serve as a negative control, obtain several data cubes from the sample. Using the hyperspectral camera, at least one is required, but more can be captured.

To increase selectivity with the negative control data cube open, click filter spectral library Under the analysis menu located on the main program toolbar, click open new file and select the spectral library created previously for the positive control as the input file, click okay. In the filter cyto viva spectral library window. Click on image and choose the output file name for the reference spectral library, and click okay.

Next, click on the file name of your negative control data cube as a source image. The software will analyze the spectral library and remove each spectrum that matches any spectrum of the negative control data cube. Obtain Data cubes from the experimental samples with the experimental data cube open.

Click on the spectral menu in the mapping methods submenu Open the spectral angle mapper. Select the name of the experimental data cube in the pop-up window, and click okay. If no file names are listed, click open new file and choose the experimental data cube.

Then click okay in the pop-up window called End Member collection, SAM, click import in the menu bar. Then select from Spectral Library file. Open the reference spectral library previously created, and click okay.

In the new popup window named Input Spectral Library, click select all items, click okay. Then right click on color and select. Apply default colors to all click select all followed by apply, and then choose the output file name and click okay.

Open the overlay menu in the data cube window and select classification. Then navigate to the output file name and click okay. In the interactive class tool window menu, open the option menu and select the merge classes to create a unified color scheme.

In order to make it easier to analyze the data using other software, select a single spectrum from the base class list. Then select all classes except unclassified to merge into the base. Then click okay.

Next, click on the base color selected and all matching spectra will be shown in that color. To obtain amatic image that will show the matching spectra over a black background, click on the unclassified colors box, which is black By default, the most significant advantage of the technique is its ability to identify metal oxide nanoparticles in histological samples by dark field microscopy and hyperspectral mapping. These images demonstrate this process using histological skin samples.

While some particles are readily obvious in the dark field, optical image, additional particles are detected using hyperspectral imaging. Shown here is porcine skin tissue in the epidermis layer, which was exposed to Illumina nanoparticles. A high contrast cluster of nanoparticles was found deposited over the stratum cornea in the dermal layer, which was exposed to silica nanoparticles.

Multiple high contrast nanoparticles can be seen deposited over the connective tissue. Finally, in the subcutaneous tissue, which was exposed to the Syria nanoparticles. Multiple high contrast dispersed nanoparticles can be seen deposited along the adipocytes nanoparticle identification using this method.

In other tissue samples such as lung, liver, spleen, kidneys, And lymph nodes is possible. Following this procedure, image analysis can be performed in order to answer additional questions such as the particle count and particle location. Certain considerations and drawbacks such as the need for highly specialized controls and the limitations on the resolution of photonic imaging must be weighed with those of conventional methods.

When selecting the most appropriate analytical techniques to investigate specific research questions, We chose to demonstrate this particular application because it has the potential to complement current and future nano toxicology research. It can also inform research regarding nanoparticle absorption, distribution, metabolism, and excretion throughout organs and tissues.