Nanotechnology offers great promise in drug delivery and medical diagnostics, but challenges remain. The accurate and reliable characterization of nanoparticles is crucial to assess their safety, efficacy, and quality in a drug substance, drug product, or other biomedical use. But the size of the nanoparticle can make this characterization difficult. The refinement of nanoscale analytical methods is of interest for nanotechnology in general and particularly for biomedical applications.
Light from the image focal plane reaches the detector, which strongly increases image contrast and slightly increases resolution. Special filters suppress the reflected laser light while enabling the Raman scattered light to be detected with a spectrometer/charged-coupled device camera combination. With this setup, a complete Raman spectrum is acquired at each image pixel, typically taking between 760 μs and 100 ms. The individual spectra are combined to form Raman images consisting of tens of thousands of spectra. From this multispectrum file, an image is generated by integrating over a certain Raman line in all spectra or by evaluating the various peak properties such as peak width, min–max analysis, or peak position. Due to the confocal arrangement, depth profiling and 3-D imaging are also possible if the sample is transparent.
"The chemical composition of nanoparticles can thus be investigated, resulting in images showing, for example, the drug distributions, core-shell compositions, or the accumulation of polymorphs," says Fischer. "In order to image the outer and inner shape of a nanoparticle in total, the size should not be smaller than the diffraction limited resolution of the microscope, which can go down to approximately 200 nm," he says. "However, smaller particles might still show a Raman signal, which allows chemical identification or localization of the particle in a specific surrounding matrix."
Atomic force microscopy
Atomic force microscopy (AFM) is a widely used tool in nanotechnology. It can be used for imaging the surface structure and organization or the orientation of the molecules that form a nanoparticle. If the particles show specific values for adhesion or stiffness, AFM can evaluate such properties on a nanometer scale. For high-resolution surface-topography imaging along with the chemical information derived from Raman imaging, a confocal Raman microscope can be transformed into an atomic force microscope by rotating the microscope turret when using a modular instrument configuration.
"AFM is used to trace the topography of samples with extremely high resolution by recording the interaction forces between the surface and a sharp tip mounted on a cantilever," says Fischer. "The sample is scanned under the tip using a piezo-driven scan-stage, and the topography is reproduced with specialized software tools, which translate this information into images. Structures below the diffraction limit can be visualized using this imaging technique." The lateral resolution (xy) is dependent on the tip radius, which is typically in the range of 5–10 nm, whereas the height resolution (z) is typically below 1 nm.
The small size of the nanoparticle creates challenges. "Mainly the difficulties arise simply due to the fact that the nanoparticles are very small, which might make it difficult to find the right sample spot," says Fischer. "Especially for AFM investigations, it is necessary to properly fix the particles on the substrate in order to prevent movement while scanning. For combined Raman/AFM investigation, easy localization of the same sample position is of importance," he adds.