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.

Date Published: 22 February 2013
PDF: 7 pages
Proc. SPIE 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX, 85891G (22 February 2013); doi: 10.1117/12.2003005

Published in SPIE Proceedings Vol. 8589:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX
Carol J. Cogswell; Thomas G. Brown; Jose-Angel Conchello; Tony Wilson, Editor(s)