Abstract
A multimodal multiphoton microscopy (MPM) is developed to acquire both two-photon microscopy (2PM) and three-photon microscopy (3PM) signals. A dual-wavelength Er-doped fiber laser is used as the light source, which provides the fundamental pulse at 1580 nm to excite third harmonic generation (THG) and the frequency-doubled pulse at 790 nm to excite intrinsic two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Due to their different contrast mechanisms, the TPEF, SHG, and THG images can acquire complementary information about tissues, including cells, collagen fibers, lipids, and interfaces, all label-free. The compact MPM imaging probe is developed using miniature objective lens and a micro-electro-mechanical scanner. Furthermore, the femtosecond laser pulses are delivered by a single mode fiber and the signals are collected by a multimode fiber, which makes the miniaturized MPM directly fiber-coupled, compact, and portable. Design considerations on using the dual excitation wavelengths are discussed. Multimodal and label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples. The miniaturized multimodal MPM is shown to have great potential for label-free imaging of thick and live tissues.
Highlights
Multiphoton microscopy (MPM) is a laser-scanning microscopy technique based on exciting and detecting nonlinear optical signals from tissues and cells [1,2,3]
Typical MPM performs two-photon microscopy (2PM) imaging, including two-photon excitation fluorescence (TPEF) [1] and second harmonic generation (SHG) [2]
TPEF is a primary signal in MPM [3], which originates from intrinsic fluorophores in tissues, such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and elastin, and exogenous fluorophores by staining the tissue with contrast agents [1]
Summary
Multiphoton microscopy (MPM) is a laser-scanning microscopy technique based on exciting and detecting nonlinear optical signals from tissues and cells [1,2,3]. The major limitation of those single wavelength laser systems in the 1.0-1.7 μm range is that intrinsic TPEF signal from tissues such as NADH and elastin cannot be excited by this long wavelength. A major challenge of the current dual-wavelength excitation sources (a laser plus OPO or two lasers) is that the whole laser system is highly complicated and bulky, which limits the clinical application of multimodal MPM. A simpler laser system that can provide dual-wavelength excitation is needed; Second, all label-free imaging of 2PM and 3PM in tissues, including TPEF, SHG, and THG is needed; Third, a compact imaging head with fiber delivery of the laser light and collection of the signals is needed. The miniaturized multimodal MPM uses a dual-wavelength EDF laser system, which provides the fundamental pulse at 1580 nm and frequency-doubled pulse at 790 nm. Our results demonstrate the capability and potential of the miniaturized multimodal MPM for future clinical applications
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