Optical Photon ReAssignment Microscopy
To enhance the resolution of a confocal laser scanning microscope the additional information of a pinhole plane image taken at every excitation scan position can be used (Sheppard 1988). This photon reassignment principle is based on the fact that the most probable position of an emitter is at half way between the nominal focus of the excitation laser and the position corresponding to the (off centre) detection position. Therefore, by reassigning the detected photons to this place, an image with enhanced detection efficiency and resolution is obtained. Here we present optical photon reassignment microscopy (OPRA) which realizes this concept in an all-optical way obviating the need for image-processing. With the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light, an image with the advantages of photon reassignment can be acquired. However, just as in computational photon reassignment, a loss in confocal sectioning performance is caused by working with relatively open pinholes. The OPRA system shares properties such as flexibility and ease of use with a confocal laser scanning microscope, and is therefore expected to be of use for future biomedical routine research.
Fig 1 The principle of OPRA. The figure shows the imaging process of one point source at different times. The fluorophore is placed at the point of origin in the sample plane (a). The emitted photons are imaged to different positions in the image plane (b) according to the excitation positions s. If the general magnification of the microscope is neglected and the intermediate magnification is m=0.5 the photons are reassigned to half the distance between the nominal excitation position s and the position of the detected photon without intermediate magnification. In normal scanning microscopy the photons are always assigned to position s. Note that the brightness changes of the green emitter caused by the variation in excitation are not shown in this scheme.
Roth et al. Optical Nanoscopy 2013, 2:5 http://www.optnano.com/content/2/1/5