Fluorescence Microscope Notes

Fluorescence Microscope the specimen is irradiated with excitation light from a fluorescence material that is suited by an excited light source Spectrom of the Fluorescence (stoke's law) Confocal microscopy -Confocal microscope was invented because of the draw backs of - fluorescence microscope The fluorescence microscope draw backs: * in fluorescence microscope there is a high intensity UV light, which couses photo bleaching in the specimen. So the image that obtained was blurred. -To solve this problem the confocal microscope was invented as a modified flourescence microscope. -with 2 major modifications: 1- it uses a laser light instead of Hg-vapor lamp. 2- images are taken with a digital Camera with a pinhole. Vallows light of only one focol plane to be focused - other lights are canceled out. on the digital camera. lights from below the focal plane. process of it: 1- laser is focased in a small region of the specimen. and creats flourescent light which is captured by the digital Camera. 2- using two rotating mirrors in my and ryll direction. - the laser light can focus on the next region. 3- The laser scons the whole surface of specimen and images of each spot is captured by the digital camera. 4_ finally a software in the computer combines all these images into one sharp image. •Used also for 3D images. Two photon microscopy built for the limitations of confocal microscopy. -it works based on Hourscent signals from the fluorescance material after 2 photon excitation by the laser light source. - photons from the excitation light are absorbed simutaneously by the floarescent material. -it Applications→1-physiology 2-Neurobiology 3-embryology 4- Cancer biology 5-tissue engineering