Rescanning Laser Scanning Confocal Microscope
Background: The laser scanning confocal microscope (LSCM) is widely used as a confocal microscopy technique for imaging fluorescent objects in a manner that removes out of focus fluorescent light and thus provides increased axial resolution and better imaging compared to a standard fluorescence microscope. A laser is used since it can generate a very intense spot of light on the specimen. A problem with this detection method is that it requires measuring a rapidly changing low level of light and reconstructing an image from this data. Photomultiplier tubes have poor efficiency at converting photons to a useable signal due to poor quantum efficiency and high noise. Technology: Researchers at the University of California have developed a LSCM design so that a confocal image of the specimen is formed and light can be detected with a camera such as cooled CCD (or other low light) cameras. The cooled CCD has a much higher quantum efficiency, much lower noise and much higher dynamic range than photomultipliers and other dynamic light detectors. These cameras also permit very long exposure times during which photons are accumulated onto the CCD. This allows imaging of very dim specimens that would be impossible to detect with a photomultiplier.
Another advantage is the simplicity of the electronic control system. The design only requires that the two mirror systems scan in synchrony. Since scanning systems with controllers are commercially available, the implementation of the invention is greatly simplified. All the imaging can be done with a standard off the shelf camera, instead of a photomultiplier. The system has the potential for low cost implementation. By rapid scanning, it would be possible to directly view the specimen through eye pieces, not possible with conventional LSCM. Application: The design developed at the University of California permits the confocal image to be acquired by a camera or viewed directly. The use of the camera such as a scientific cooled CCD permits substantially greater sensitivity, increased signal to noise ration and larger dynamic range compared to photomultipliers. It also simplifies image acquisition since the image is generated directly and unlike the conventional LSCM which must reconstruct the image from time varying data.
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