Miniaturized multimodal multiphoton microscope for simultaneous two-photon and three-photon imaging with a dual-wavelength Er-doped fiber laser

Abstract

A multimodal multiphoton microscopy (MPM) is developed to acquire both twophoton 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.

Single-cavity dual-wavelength all-fiber femtosecond laser for multimodal multiphoton microscopy

“…Even though the three wavelengths are generated from a single cavity which reduces the cost, the laser source is not an all-fiber design which makes it vulnerable to alignment and vibration. Similar approach has been recently presented in [18,19].…”

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

“…Additionally, understanding the biological relevance of these biomarkers and their associated regulators is equally important in furthering the therapeutics and diagnostics of CAVD, a disease with no drug-based therapies, or early diagnostic tests (4-7). Advancements in probe design for accessibility and challenges associated with imaging moving objects with multiple confounding autofluorescent and absorbing sources must be overcome for the successful adoption of MPM techniques and metrics for clinical imaging of aortic valves (28,97,(101)(102)(103)(104)(105).…”

“…MPM-based intravital imaging of the ventricular wall has been performed (15,16,99,100); however, in vivo imaging of valves faces challenges due to accessibility, tissue movement, and blood flow (28,97,(101)(102)(103)(104). Limitations of tissue accessibility are being addressed by enhancing flexibility and miniaturization of microendoscopy tools, which will help facilitate preclinical and clinical translation of MPM (28,(101)(102)(103)105). Work is also focused on developing strategies to overcome motion-based artifacts introduced by heartbeat or physiological geometry changes (104).…”

Biomedical Optical Sensors

“…Even though the three wavelengths are generated from a single cavity which reduces the cost, the laser source is not an all-fiber design which makes it vulnerable to alignment and vibration. A similar approach has been recently presented in [61].…”