Darkfield microscopy

Darkfield microscopy is a special imaging technique to render transparent and un- stained objects visible, particular if their size is below the diffraction limit. It is also employed investigating plasmonic properties of metal nanoparticles [62–64]. Only light scattering off optical discontinuities such as media boundaries or edges con- tributes to the image, unscattered light is not collected by the viewing objective. The resulting image shows the bright structures of the object of interest on a con- trasting dark background. Darkfield imaging is achieved either in transmission or reflection. Reflective darkfield microscopy requires specialised objectives with outer and inner beam paths as well as special darkfield cubes (mirrors with circular clear- ance in the centre) in the illumination beam path. We used transmission darkfield imaging as pictured in Fig 3.16.

In a standard darkfield configuration, an aperture blocking the central part of the illumination is placed in the beam path. The condenser focuses a hollow cone of light into the sample chamber. With no sample present the light rays miss the viewing objective and the field of view remains dark. Inserting a sample at the tip of the inverted hollow light cone scatters the incident light at the sample. The scattered light is collected by the viewing objective while the oblique illumination of the hollow cone keeps missing the objective. We used an oil immersion darkfield condenser from Nikon (NA 1.20-1.43) for our experiment. There are more sophisti-

laser line mirror microscope objective lens 2 lens 1 digital colour CCD camera aperture darkfield condenser sample laser halogen lamp f2 _{4 m polymer spheres} Nikon, 40x, NA 0.65 80nm silver spheres Nikon, 100x oil, NA 1.3

Figure 3.16: Left - The diagram pictures the illumination beam path for darkfield mi-

croscopy in transmission. The central part of the light beam is blocked before it enters the darkfield condenser. The so-created hollow cone of light is then focussed into the sam- ple chamber where it scatters off the metal nanoparticles. The scattered light enters the viewing objective while the direct illumination misses it. We use the viewing objective as trapping objective at the same time. Right- We present two sample pictures acquired with our darkfield setup. The 4µm polymer spheres are imaged with a 40x objective (Nikon Plan Apo, NA 0.65). The 80nm silver spheres are imaged with a 100x objective (Nikon CFI Plan Fluor, oil, NA 0.7-1.3) which we used for trapping later on.

cated systems available to improve the quality of the image, in particular for high NA applications [65, 66].

Regardless of the darkfield condenser used, it is critical to match the condenser and the viewing objective. The best darkfield condenser is ineffective if the NA of the viewing objective is larger than the NA of the darkfield condenser. The direct illumination is then collected by the viewing objective and no dark background can be achieved. To optically trap metal nanoparticles we use a trapping objective with an NA as large as possible in order to focus the trapping laser as tightly as possible. The trapping objective is necessarily the viewing objective in a darkfield configuration as the darkfield condenser block would disturb the laser beam path. Keeping all these factors in mind, we find that despite aiming for the largest NA possible for our trapping/viewing objective we have to keep it smaller then the NA of the darkfield condenser.

In our experiment we used a Nikon oil immersion darkfield condenser (NA 1.20- 1.43) and an oil immersion Nikon CFI Plan Fluor 100x objective (NA 0.7-1.3 ad- justable). There have been trapping studies with lower NA objectives, but these only investigated optical trapping of metal nanoparticles in two dimensions as real three dimensional trapping cannot be achieved with low NA objectives [9]. We achieved

3D trapping of metal nanoparticles while imaging with the above described darkfield illumination (as pictured in Fig. 3.16). However, we do not consider this kind of darkfield illumination practical to use in conjunction with optical trapping, mainly because of two reasons. First, the sample is enclosed by two oil immersion objec- tives. That renders a sample exchange rather delicate and introduces additional aberrations of the oil films (the oil for the darkfield condenser was very viscous). Second, the only way to add a quadrant photodiode to this system for position de- tection (see Sec. 3.4.2) is in back reflection. It turns out that this type of particle detection is already difficult enough for metal nanoparticles in a forward scattering configuration and practically impossible in a back scattering setup.

We thus proceeded in a different way to image metal nanoparticles in an optical trap. The best way of illuminating metal nanoparticles while trapping them turned out to be the brightfield episcopic imaging system we described in the previous sec- tion. Brightness and contrast are adjusted as described before with the condenser and field aperture of the K¨ohler illumination beam path. Including K¨ohler illu- mination in our setup ensures the best possible illumination providing an evenly illuminated field of view as well as best possible contrast and resolution.