Zeiss LSM 510 Operating Manual

Also see for LSM 510 Inverted: Quick guide Operating manual User guide User manual Brief Operating Manual

INTRODUCTION TO LASER SCANNING MICROSCOPYLSM 510 Principle of Laser Scanning MicroscopyB 40-051 e 07/98 3-33 INTRODUCTION TO LASER SCANNING MICROSCOPY3.1 Principle of Laser Scanning MicroscopyTo yield information on their inner structure by conventional transmitted-light microscopy, specimenshave to be very thin and translucent; otherwise image definition will be poor. In many cases it is aproblem to satisfy these requirements.The essential considerations have led to trailblazing changes in conventional microscopy and supplied asuccessful solution to the above problem. Unlike the practice of even illumination in conventional microscopy, the LSM technique projects thelight of a point light source (a laser) through a high-NA objective onto a certain object plane ofinterest as a nearly diffraction-limited focus. However, if not for another "trick", the stray lightproduced outside the object plane, or the fluorescence of fluorescent specimens, would disturb thein-focus image of object point of interest, resulting in a blurred image of poor contrast. The problemis therefore, how to capture only the light coming immediately from the object point in focus, whileobstructing the light coming from out-of-focus areas of the specimen. The light reflected, or the fluorescence lightproduced, at the focus of the high-NA objectiveis projected onto a variable pinhole diaphragmby the same objective and a tube lens. Thefocus inside the specimen and the pinhole aresituated at optically conjugate points (confocalimaging). The decisive advantage of thisarrangement is the fact that essentially no otherlight than that coming from the object plane ofinterest can pass the narrow pinhole and beregistered by a detector. Unwanted lightcoming from other specimen areas is focusedoutside the pinhole, which passes only a smallfraction of it. The smaller the pinhole, the lessstray light or fluorescence from out-of-focusareas will get on the detector. The image pointthus generated is largely free from blur causedby unwanted light.Fig 3-1 Principle of confocal imaging