We propose a new method for three-dimensional fluorescence imaging without depth scanning that we refer to as the
dual detection confocal fluorescence microscopy (DDCFM). Compared to conventional beam-scanning confocal
fluorescence microscopy, where the focal spot must be scanned either optically or mechanically to collect a three-dimensional
images, DDCFM is able to obtain depth information without depth scanning. DDCFM utilizes two photo
multiplier tubes (PMTs) in the confocal detection system. The emitted fluorescence is divided by the beam splitter and
received by the two PMTs through pinholes with different size. Each PMT signal generates different axial response
curve according to the pinhole diameter, which decides stiffness of the curve. Since the PMT signal is determined by the
intensity of the fluorescent emitter and the distance from the focal point, we can acquire depth position of a fluorescent
emitter by comparing two intensity signals from the PMTs. Since the depth information can be obtained by a single
excitation without depth scanning, DDCFM has many advantages. The measurement time is dramatically reduced for
volume imaging. Also, photo-bleaching and photo-toxicity can be minimized. The system can be easily miniaturized
because no mechanical depth scan is needed. Here, we demonstrate the feasibility of the proposed method by phantom
study using fluorescent beads.
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Dong-Ryoung Lee ; Young-Duk Kim ; Hyeong-Jun Jeong ; Jong-Min Lee ; Dae-Gab Gweon, et al.
Dual detection confocal fluorescence microscopy: depth imaging without depth scanning
", Proc. SPIE 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX, 85891G (February 22, 2013); doi:10.1117/12.2003005; http://dx.doi.org/10.1117/12.2003005