A custom-built two-photon microscope based on a mode-locked Yb3+ doped fiber laser

Dong Uk Kim; Hoseong Song; Woosub Song III; Hyuk-Sang Kwon; Dug Yong Kim

doi:10.1117/12.907648

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2 February 2012 A custom-built two-photon microscope based on a mode-locked Yb³⁺ doped fiber laser

Dong Uk Kim, Hoseong Song, Woosub Song III, Hyuk-Sang Kwon, Dug Yong Kim

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Proceedings Volume 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX; 82271F (2012) https://doi.org/10.1117/12.907648
Event: SPIE BiOS, 2012, San Francisco, California, United States

Abstract

Two-photon microscopy is a very attractive tool for the study of the three-dimensional (3D) and dynamic processes in cells and tissues. One of the feasible constructions of two-photon microscopy is the combination a confocal laser scanning microscope and a mode-locked Ti:sapphire laser. Even though this approach is the simplest and fastest implementation, this system is highly cost-intensive and considerably difficult in modification. Many researcher therefore decide to build a more cost-effective and flexible system with a self-developed software for operation and data acquisition. We present a custom-built two-photon microscope based on a mode-locked Yb³⁺ doped fiber laser and demonstrate two-photon fluorescence imaging of biological specimens. The mode-locked fiber laser at 1060 nm delivers 320 fs laser pulses at a frequency of 36 MHz up to average power of 80 mW. The excitation at 1060 nm can be more suitable in thick, turbid samples for 3D image construction as well as cell viability. The system can simply accomplish confocal and two-photon mode by an additional optical coupler that allows conventional laser source to transfer to the scanning head. The normal frame rate is 1 frames/s for 400 x 400 pixel images. The measured full width at half maximum resolutions were about 0.44 μm laterally and 1.34 μm axially. A multi-color stained convallaria, rat basophilic leukemia cells and a rat brain tissue were observed by two-photon fluorescence imaging in our system.

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Dong Uk Kim, Hoseong Song, Woosub Song III, Hyuk-Sang Kwon, and Dug Yong Kim "A custom-built two-photon microscope based on a mode-locked Yb³⁺ doped fiber laser", Proc. SPIE 8227, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XIX, 82271F (2 February 2012); https://doi.org/10.1117/12.907648

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