Design and Development of Nonlinear Optical Microscope System: Simple Implementation with epi-Illumination Platform

Abstract

During the research using fluorescence-tagged or auto-fluorescence molecules, meaningful information is often buried deep inside the tissue, not its surface. Therefore, especially in the field of biomedical imaging, acquiring optically sectioned images from deep inside the tissue is very important. As well know already, confocal laser scanning microscopy (the most well-known optical sectioning microscopy) gives axially-resolved fluorescence information using the physical background blocking component called pinhole. However, the axial range of imaging is practically limited due to such optical phenomena as the light scattered and absorbed in the tissue. However, nonlinear optical microscopy (e.g. Multiphoton microscopy, harmonic generation microscopy, coherent anti-Stokes Raman spectroscopy) realized by the development of ultrafast light sources has been used for visualizing various tissues, especially in vivo, because of their low sensitivity to the limitation caused by the scattering and the absorption of light. Although nonlinear optical microscopy gives deep tissue image, it is not easy for many researcher to build customized nonlinear system. Here, we introduce an easy and simple way designing and developing such nonlinear optical microscope with upright or inverted epi-illumination platform using commercial optical components only.