Dual detection confocal fluorescence microscopy: depth imaging without depth scanning

Dong-Ryoung Lee; Young-Duk Kim; Hyeong-Jun Jeong; Jong-Min Lee; Dae-Gab Gweon; Hongki Yoo

doi:10.1117/12.2003005

22 February 2013 Dual detection confocal fluorescence microscopy: depth imaging without depth scanning

Dong-Ryoung Lee, Young-Duk Kim, Hyeong-Jun Jeong, Jong-Min Lee, Dae-Gab Gweon, Hongki Yoo

Proceedings Volume 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX; 85891G (2013) https://doi.org/10.1117/12.2003005
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

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.

Citation Download Citation

Dong-Ryoung Lee, Young-Duk Kim, Hyeong-Jun Jeong, Jong-Min Lee, Dae-Gab Gweon, and Hongki Yoo "Dual detection confocal fluorescence microscopy: depth imaging without depth scanning", Proc. SPIE 8589, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XX, 85891G (22 February 2013); https://doi.org/10.1117/12.2003005

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KEYWORDS

Confocal microscopy

Luminescence

Microscopy

Quantum efficiency

3D image processing

Fluorescence spectroscopy

Mirrors

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