DIC microscopy is superior to dark field at imaging plasmonic nanoparticles in complex environments. However, DIC also requires greater knowledge and skill to obtain reproducible results. DIC microscopy provides both high lateral resolution and shallow depth of field.
Both of these traits are extremely important when monitoring nanoparticles within cells or in complex environments. To begin, use a scribing pen to place a shallow and short scratch mark onto the center of each glass coverslip, and then clean the glass as described in the accompanying text protocol. Next, use a micropipette to remove 100 microliters of a 05-milligram-per-milliliter gold nanoparticle solution from its original storage container, and eject the solution into a 1.5-milliliter centrifuge tube.
Centrifuge the sample for 10 minutes at 6, 000 times gravity. When finished, remove the supernatant with a micropipette to get rid of the excess surfactant from the nanoparticle solution. Next, add 100 microliters of ultra-pure water to the centrifuge tube.
Briefly vortex the sample to break up the pellet, and then sonicate it for 20 minutes immediately afterwards to fully resuspend the nanoparticle aggregates. Using a micropipette, dropcast six microliters of nanoparticle solution onto the cleaned and scratched glass coverslip. Then carefully flip the coverslip and place it on top of a second larger piece of microscope glass.
The drop should quickly spread out evenly between the two pieces of glass. Take care to avoid getting air bubbles trapped between the two pieces of glass. Use a narrow line of nail polish to seal off the edges of the coverslip to prevent evaporation of the liquid.
Place the sample onto the microscope stage, and align the objective and condenser of the microscope. Adjust the focus to find the focal plane with the sample on it and locate the scratch mark created earlier. Focus on it and fine-tune the focus until the nanoparticles come into view.
To determine the accurate placement of the condenser, utilize the Kohler illumination method by starting with the 20x objective, and then moving to high magnifications such as 80 or 100x. Next, select a region of interest within the sample for imaging. Center the region in the camera's field of view, and adjust the focus as necessary.
If the microscope has the de Senarmont design, start with the polarizer set near to maximum background extinction and gradually rotate the polarizer towards decreasing background extinction. The background intensity will gradually increase. The ideal setting is achieved when the nanoparticles reach their greatest intensity difference compared to the local background average.
For plasmonic nanoparticles, greatest contrast is typically achieved with a relatively dark background at or near the maximum background extinction. Turn off room lighting to prevent stray illumination from interacting with the process. Put into place a 10-nanometer full-width at half-maximum band-pass filter with its central wavelength co-located with the main localized surface plasmon resonance wavelength in order to view the region of interest.
Next, adjust the lamp intensity or exposure time until the background level is at 5%to 40%of the camera's maximum capacity level. No objects within the region of interest should exhibit signal intensities that exceed 90%of the camera's maximum intensity level. Image the sample with a series of band-pass filters that each has a full width at half-maximum of 10 nanometers, and that as a whole enable imaging across the entire wavelength range of interest.
Ensure that the background intensity in the images remains within 5%of one another by adjusting the exposure time and not the lamp power. After switching filters, refocus the sample before capturing images. Save the images as uncompressed TIFF files and/or in the software's native file format in order to preserve all relevant information.
To begin analysis, open the image with ImageJ. Use the rectangle tool to draw a rectangle around the main region of interest. Then on the toolbar, select image, go to zoom, and select to selection.
The imaging window will zoom in on the selected area. Next, select image, go to adjust, and select brightness/contrast. To improve the view of the sample region, adjust the minimum, maximum, brightness, and contrast of the image.
These adjustments do not alter the scientific data, they merely enable better visibility of the sample region. Now, use the rectangle tool again and draw a box around the first nanoparticle to be measured. The box should be only slightly larger than the nanoparticle's airy disc.
Once selected, go to analyze, and select measure. A new window appears that reports the minimum, maximum, and mean intensities for the pixels located inside of the selected box. Retaining the original size of the box, drag the box to an area immediately adjacent to the particle where the background contrast is relatively even, and no particles or contaminants are present.
Use the measure tool again to determine the mean intensity for the background area. Repeat this process for each particle in all of the images in the series. Then export the data to a spreadsheet to calculate the contrast or intensity of each particle across all wavelengths and angles.
Enter in the following equation to calculate each particle's contrast. Finally, graph the spectral profile at a given nanoparticle position by plotting data with the wavelength along the X-axis and the contrast or intensity along the Y-axis. To determine the optimal imaging setting, these gold nanospheres were imaged at several polarizer settings.
At zero degrees, the particles appear mostly white with a dark stripe running across their midsection. This is indicative of cross-polarization for nanosphere samples. When the polarizer is rotated to different angles, the particles cast dark shadows towards one corner.
However, the particle contrast values change dramatically. For this sample, the optimal imaging setting is with a polarizer shift of 10 degrees. Here the wavelength is plotted against contrast of gold nanospheres.
Each data point represents an average of 20 nanospheres for each particle diameter. The peak contrast for each particle shifts slightly depending on its size. The intensity and rotation is even more important with anisotropic shapes such as this gold nanorod.
Here the wavelength is plotted against intensity at two different polarizer settings. In each case, the bright side's intensity is much stronger than the dark side's. The intensity profile of a single gold nanorod at its localized surface plasmon resonance wavelength during rotation of the stage shows a large intensity difference between the dark and bright sides as the sample is rotated through 180 degrees.
Sample imaging is extremely important. It requires both diligence and patience on the part of the microscopist. If you are performing an experiment that requires reimaging the sample over a series of days, then landmarks such as the scratch marks are critical in relocating your region of interest.
DIC microscopy is growing in interest by researchers who study nanoparticles, especially in dynamic and cellular environments. Because DIC offers optimal spatial resolution, especially in complex sampling environments.
