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.
Raman spectroscopy is one example of an analytical method that can be employed on a nanoscale. Raman spectroscopy measures
the vibrational frequencies of various parts of a molecule. These frequencies depend on the bond strength, the mass of the
bound atoms, and other factors, including intermolecular interactions and the crystalline arrangement of solids (1). While
infrared (IR) spectroscopy is based on illuminating a sample with a broad range of wavelengths of IR light and measuring which
are absorbed, a Raman spectrum is obtained by illuminating a sample with a single wavelength of light from a laser and collecting
and analyzing the resulting scattered light. Raman spectroscopy is a nondestructive method and can be used in identifying
and testing raw materials, in-process materials, polymorphs as well as for assessing content uniformity and small particulates
(IMAGE: PASIEKA/SPL, SCIENCE PHOTO LIBRARY, GETTY IMAGES)
In nanoparticle analysis, Raman spectroscopy is used with confocal microscopy, an optical imaging technique that increases
optical resolution. "By integrating a Raman spectrometer within a confocal microscope setup, Raman imaging with a spatial
resolution down to 200 nm laterally and 500 nm vertically can be achieved using visible-light excitation," explains Harald
Fischer, marketing director of WITec GmbH (Ulm, Germany), a manufacturer of high-resolution optical and scanning-probe microscopy
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.