Probe microscopy: scanning below the cell surface.

M ore and more nanoparticles are used in advanced materials, medical imaging and drug delivery, and their industrial scale production is just around the corner. The flip side of this is the risk associated with human exposure to nanoparticles, whether through unintentional inhalation or deliberate injection and ingestion. Detailed toxicity studies are needed to ensure the safe and successful use of nanoparticles, but numerous challenges exist1. In particular, visualizing the way that nanoparticles enter cells is an area that deserves attention. On page 501 of this issue, Ali Passian and colleagues at the Oak Ridge National Laboratory, the University of Tennessee and Northwestern University report striking images of nanoparticles inside cells obtained by purely mechanical means2. Their method does not require using any external labels such as fluorescent or radioactive molecules to tag the cells or particles. This makes the approach simple and promises throughput with minimal sample preparation. Considering the large variety of nanoparticles and cells that exist, these attributes will be immensely useful in determining the fate of nanoparticles that come into contact with biological organisms. The use of optical microscopes to image particles much smaller than the wavelength of light has proven to be difficult, but it is possible. Diffraction of light waves results in blurry images that have little contrast for objects smaller than the wavelength of light. A clever method to overcome the diffraction limit is to bring the optical sensor close to the sample. If the distance between the sensor and the sample is smaller than the wavelength of light then high resolution images can be generated. This powerful imaging method is generally termed as near-field scanning optical microscopy3. The advantages of near-field optical microscopy are balanced by the geometrical constraints on the sample. Unfortunately, objects that are buried several micrometres below the surface, such as nanoparticles that are inside cells, cannot take advantage of near-field optical microscopy. The analogy between ultrasonic waves and light waves is used in the study by Passian and co-workers. Ultrasonic waves are high frequency mechanical vibrations and, like optical waves, they can be used to generate images of samples including cells4. The concept of near-field imaging is also applicable to ultrasonic waves5, but with a significant advantage. The ultrasonic wavelengths used for imaging can be as long as a millimetre, which is much longer than the wavelength of visible light. Nanoparticles that are below the cell surface are still in the ultrasonic near field, so high-resolution images can be generated by near-field techniques. Passian and colleagues applied this technique, known as scanning near-field ultrasonic holography6, to image nanoparticles inside cells. In the experiments, they aspirated mice with single-walled carbon nanohorn particles and isolated the macrophages (cells that engulf foreign material) from the lungs and red blood cells for imaging. The cells were placed on a substrate that was vibrated at ultrasonic frequencies of around 4 MHz (Fig.1). As the vibrations propagated through the cells, depending on the objects in their paths, different regions on the wave fronts accumulated different delays or phase shifts. Hence, the original wave front is disturbed after interacting with the nanoparticles. A frequency of 4 MHz corresponds to a wavelength of around a quarter of a millimetre, which is more than a thousand times longer than the dimensions of nanoparticles. This means that the phase shifts due to nanoparticles can be extremely small. Therefore a sensitive detection mechanism is needed. The team uses the sharp tip of an atomic force microscope to measure the disturbances or delays in the wave fronts by vibrating the microscope cantilever at a slightly different frequency compared with the vibrations emanating from the substrate (much like the way an FM radio receiver converts high frequency electromagnetic waves into much lower Conventional atomic force microscopy probes only the surface of specimens. A related technique called scanning near-field ultrasonic holography can now image nanoparticles buried below the surfaces of cells, which could prove useful in nanotoxicology. proBe MiCroSCopy