Multiphoton Imaging Systems

Abstract

The advent of confocal laser scanning microscopy revolutionized the field of fluorescent imaging. It allowed researches to obtain high resolution, real time, three-dimensional images of cells that were previously unobtainable using traditional imaging modalities [1]. This development helped accelerate research in many of the life science branches due to its ability to achieve superior optical sectioning and signal to noise over conventional wide field fluorescence approaches. However, traditional confocal laser scanning microscopy has several drawbacks. First, the use of a pinhole, although effective, does not entirely eliminate scattered light from out of focus planes from reaching the photodetector. Second, the wavelengths used in excitation, typically in the UV and near UV range, tend to be particularly harmful to living specimens [2]. Finally, because of wavelengths used, the penetration depth of this modality is limited typically to about 100 microns. These drawbacks led researchers to search for a modality that addressed these issues. This led to the research and development of multiphoton laser scanning microscopy.