Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits
of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization
microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular
organizations. A combination of these methods seems particularly attractive as it allows adapting to the required
resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures.
SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly
resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the
distribution of one protein and its associated structure can be viewed in the context of others. Since all common
fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly
resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to
count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked
simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the
axial direction as well. These applications start already to have an impact on our view how a cell is organized and how
different proteins contribute to its make-up.
The diffraction limit in traditional fluorescence microscopy (approximately 200 and 600 nanometers in lateral and axial
directions, respectively) has restricted the applications in
bio-medical research. However, over the last 10 years various
techniques have emerged to overcome this limit. Each of these techniques has its own characteristics that influence its
application in biology. This paper will show how two of the techniques, Structured Illumination Microscopy (SIM) and
PhotoActivated Localization Microscopy (PALM), complement each other in imaging of biological samples beyond the
resolution of classical widefield fluorescence microscopy. As a reference the properties of two well known standard
imaging techniques in this field, confocal Laser Scanning Microscopy (LSM) and Total Internal Reflection (TIRF)
microscopy, are compared to the properties of the two high resolution techniques.
Combined SIM/PALM imaging allows the extremely accurate localization of individual molecules within the context of
various fluorescent structures already resolved in 3D with a resolution of up to 100nm using SIM. Such a combined
system provides the biologist with an unprecedented view of the
sub-cellular organization of life.