Chulmin Joo

Multiphoton microscopy with near infrared contrast agents

While multiphoton microscopy (MPM) has been performed with a wide range of excitation wavelengths, fluorescence emission has been limited to the visible spectrum. We introduce a paradigm for MPM of near-infrared (NIR) fluorescent molecular probes via nonlinear excitation at 1550 nm. This all-NIR system expands the range of available MPM fluorophores, virtually eliminates background autofluorescence, and allows for use of fiber-based, turnkey ultrafast lasers developed for telecommunications.

Two-photon optical properties of near-infrared dyes at 1.55 μm excitation.

Two-photon (2P) optical properties of cyanine dyes were evaluated using a 2P fluorescence spectrophotometer with 1.55 μm excitation. We report the 2P characteristics of common NIR polymethine dyes, including their 2P action cross sections and the 2P excited fluorescence lifetime. One of the dyes, DTTC, showed the highest 2P action cross-section (∼103 ± 19 GM) and relatively high 2P excited fluorescence lifetime and can be used as a scaffold for the synthesis of 2P molecular imaging probes. The 2P action cross-section of DTTC and the lifetime were also highly sensitive to the solvent polarity, providing other additional parameters for its use in optical imaging and the mechanism for probing environmental factors. Overall, this study demonstrated the quantitative measurement of 2P properties of NIR dyes and established the foundation for designing molecular probes for 2P imaging applications in the NIR region.

Autoconfocal transmission microscopy based on two-photon-induced photocurrent of Si photodiodes

We describe a simple, self-aligned confocal transmission microscopy technique based on two-photon-induced photocurrents of silicon photodiodes. Silicon detectors produce photocurrents in quadratic dependence on incident intensity under the pulsed illumination of light with wavelengths longer than 1.2 microm. We exploit this nonlinear process to reject out-of-focus background and perform depth-sectioning microscopic imaging. We demonstrate a comparable background rejection capability of the technique to linear confocal detection and present three-dimensional imaging in biological specimens.

Multimodal Nonlinear Microscopy at 1.5 μm

We describe multimodal nonlinear microscopy using a compact, turnkey femtosecond fiber laser at 1.5 μm. The system allows for multiplexed detection of near infrared and visible contrast agents through two-and three-photon excitation fluorescence microscopy as well as structural imaging viaauto-confocal microscopy (ACM). This platform expands the available emission spectrum for multiphoton microscopy, enables simultaneous structural and functional imaging, and offers advantages in penetration depth, contrast, and simplicity as compared to conventional MPM near 800 nm excitation.