Confocal Laser Scanning Microscopy (CLSM)

Abstract

Confocal microscopy, which was patented by Dr. Marvin Minsky [157] at Harvard University in 1957, dramatically improves the optical imaging resolution to unprecedented levels. The unique feature of confocal microscopy is its ability to deliver extremely thin, in-focus images by a true means of depth-wise optical slicing. This also allows for the gathering of three-dimensional reconstructed information from the line-of-sight depth-wise resolved imaging without the need for any invasive slicing of the specimens [158-160]. The confocal microscopy has been widely used in the biological, pharmaceutical, medical, and material science research areas and is often associated with laser-induced fluorescence (LIF) imaging, which allows microstructures to be more clearly visible [161,162]. Fluidic applications, however, were not realized until the optically sliced microscopic particle imaging velocimetry (PIV) was developed by Park et al. of the author’s group [163]. This innovation is named “confocal laser scanning microscopic-PIV” (CLSM-PIV). The basic principles of confocal imaging are discussed concisely in Section 4.1. General discussions on microscopic imaging resolutions are presented in Section 4.2. In-depth and mathematical discussions for the confocal imaging resolutions and optical slicing are presented in Sections 4.3 and 4.4, respectively. Finally, the CLSM-PIV system is discussed in Section 4.5, and its applications to flow measurements are presented in Section 4.6.