Re-scan confocal microscopy

One of the instruments that gave insight in the morphology and function of cellular components is the optical microscope. Nowadays, optical microscopy in biomedical applications is commonly combined with fluorescence. One fundamental limit in the possibility to distinguish small structures in the sample in fluorescence microscopy is limited by the diffraction of light. Objects that are closer together than the diffraction limit cannot be distinguished. This thesis describes a new super-resolution technique, Re-scan Confocal Microscopy (RCM) which has 1.4 times higher resolution than the diffraction limit. RCM uses a sensitive camera for detection, and therefore combines high resolution with high detection sensitivity. This thesis describes the method in detail and the theory behind the technology. We present the method for the first time, show the proof of principle and characterize its imaging properties. RCM can be used for a wide range of biomedical applications. RCM has been tuned to image multicolor samples and to perform functional studies for example in HeLa cells, yeast cells and neurons. The RCM technique has been combined with spatially-controlled illumination (SCIM) in order to minimize the illumination of the sample for reduction of phototoxicity. In this thesis it is proven that the RCM microscope is a valid alternative to standard confocal microscopy for a variety of biomedical applications where high resolution is required in combination with high sensitivity.