One major advantage of multiphoton microscopy (MPM) is that it can image below tissue surface and produce a stack of images showing sample structure at various depths. A miniature objective with depth scanning capability is needed for MPM endoscopy. Spherical aberration may be induced when changing the focusing depth during multiphoton microscope depth scanning, thus limiting the range over which images may be acquired. A specially designed miniature objective that minimizes spherical aberration across large range of focusing depths is presented. Simulations show that the 0.53 numerical aperture design can achieve on-axis diffraction limited focusing in water for depths from 0 μm to just over 1400 μm and a diffraction limited field of view of up to 290 μm for a 790 nm laser. In experiment, our multiphoton microscope demonstrates a field of view of 64 μm by 100 μm and a depth scanning range of 440 μm, limited by the scanning hardware. Depth scanning capability is confirmed by imaging 0.1 μm diameter fluorescent beads across the 440 μm range. Biological samples to a depth of 150 μm are imaged using the custom objective; the imaging depth is mainly limited by the absorption and scattering of the sample.
We report on a multimodal multiphoton microscopy (MPM) system with depth scanning. An Er-doped fiber laser with 1580 nm and 790 nm output provides the dual-wavelength multimodal capability and a shape-memory-alloy (SMA) based depth-scanning objective enables the depth scanning. Image stacks combining two-photon-excitation-fluorescence (TPEF), second-harmonic-generation (SHG), and third-harmonic-generation (THG) signals have been acquired on animal samples from the surface to over 200 μm underneath.
Multimodal multiphoton microscopy (MPM) can provide fast, label-free, non-invasive examination of cells, extracellular matrix, and lipids. Two-photon microscopy (2PM) can detect second harmonic generation (SHG) from fibrillar collagen and striated muscle myosin, whereas two-photon excitation fluorescence (2PEF) can detect intrinsic fluorophores such as NADH from cells. Meanwhile, three-photon microscopy (3PM) can detect third harmonic generation (THG) from lipids and tissue interfaces. We have developed a miniaturized multimodal multiphoton system which can perform label-free two-photon and three-photon imaging. An Er-doped fiber laser delivers fundamental pulses at 1580 nm and 80 fs for exciting THG. SHG and 2PEF are excited at 790 nm via the frequency doubling of 1580 nm pulses. For clinical applications, a compact probe is being developed with single-mode fiber for delivering the femtosecond excitation pulses and multi-mode fiber for collecting the MPM signals. A MEMS mirror performs lateral scanning at up to 4 frames/s. For objective lenses, a miniature aspherical lens (NA=0.64) is compared with a gradient index microobjective (NA=0.8). Shape memory alloy actuator used in smartphone cameras is evaluated for shifting the focal plane to acquire Z-stacks for 3D tissue imaging. High-resolution SHG, 2PEF, and THG images are acquired from biological tissues and show that multimodal MPM endomicroscopy has great potential for clinical applications as an alternative to histology.
Skin cancer is the most common form of cancer in North America, and melanoma is the most deadly form of skin cancer. Roughness assessment of epidermis has been shown to be valuable in detecting potential skin neoplasia. However, the existing roughness assessment techniques cannot also provide volumetric information. For greater insight, we propose polarization sensitive optical coherence tomography (PS-OCT) for skin assessment. The intensity channel of OCT visualizes the layered structure and surface roughness profile of skin in 3D. Furthermore, PS-OCT can simultaneously conduct polarization related measurements such as the degree of polarization uniformity (DOPU) in a separate imaging channel. Skin phantoms of different surface roughness ranging from 1 to 68 μm have been studied. It was observed that for rougher surfaces, the roughness can be quantified from the surface profile visible in the intensity channel. In smoother surfaces for which the profile is not sensitive, the DOPU decreases with roughness in a quantifiable correlation. The contrast in the DOPU channel is sensitive to polarization and phase fluctuations. Smoother surfaces tend to maintain the polarization state, whereas the height differences in a rougher surface contribute to larger phase shifts between light waves within the coherence volume, leading to greater depolarization. PS-OCT was also applied to in vivo imaging of human skin. The skin at the palm edge shows lower DOPU compared to the skin on the back of the hand, an indication of greater polarization state modification caused by skin roughness. PS-OCT can provide a comprehensive evaluation of skin, which has great potential for detecting melanoma.
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