Image Fusion Methods for Confocal Scanning Laser Microscopy Experimented on Images of Photonic Quantum Ring Laser Devices

Abstract

Confocal Scanning Laser Microscopy (CSLM) represents one of the most important advances in optical microscopy of the last decades. It is widely accepted that the confocal microscope was invented by Marvin Minsky, who filed a patent in 1957 (Minsky, 1957). However, at that time such a system was very difficult, if not impossible, to implement, due to the unavailability of required laser sources, sensitve photomultipliers or computer image storage possibilities. A laser scanning microscope using mechanical object scanning was developed in Oxford in 1975, and a review of this work was later published (Sheppard, 1990). The Oxford microscope was the first commercial confocal microscope. Other important contributors to this era of the development of confocal microscopy were Brakenhoff (Brakenhoff et al., 1979) and Cox (Cox, 1984). The architecture of a CSLM system provides the possibility to acquire images representing optical sections on a sample’s volume. In order to achieve this, in a CSLM system an excitation source emits coherent light (laser) which is scanned across the sample surface. As it reaches the sample the light is reflected towards a detector, in reflection work mode, the same optical path being used as well in fluorescence work mode. While in conventional microscopy, the detector is subjected to light which is reflected by out of focus planes, resulting in out-of-focus blur being contained in the final image, the architecture of a CSLM system helps avoid this situation. In order to acquire images corresponding to certain optical sections, a confocal aperture (usually known as pinhole) is situated in front of the detector. More precisely, the pinhole is placed in a plane conjugate to the intermediate image plane and, thus, to the object plane of the microscope. As a result, only light reflected from the focal plane reaches the detector, out-of-focus light being blocked by the pinhole (Fig. 1). The dimension of the pinhole is variable and together with the wavelength which is being used and the numerical aperture of the objective, dictates the thickness of the volume which contributes to the collected image (Shepard et. al., 1997; Wilson, 2001). In the case of CSLM systems, the detector is a photo multiplier tube (PMT), which presents a wide dynamic range and has high photon sensitivity suitable for detecting both strong and weak signal at a very quick refresh rates, in a time range of nano-seconds. The PMT detects light and converts photon hits into analogue electron flow as electrons leave the