Conventional multiphoton microscopy uses periodically pulsed sources as excitation and the sample is illuminated uniformly by the laser. While necessary for structural imaging, monitoring dynamic biological functions such as neuronal activity in the brain typically only requires imaging of the region of interest (ROI), e.g., the neurons. The adaptive excitation source enables imaging of the region of interest only. It reduces the requirement for the output power of the excitation source (by at least an order of magnitude) and simultaneously reduces the excitation power to the sample for obtaining the necessary information (e.g., neuronal activity). We demonstrate three-photon imaging of brain activity in awake transgenic mice (jRGECO1a), with highest speed (30 frames/s), large field-of-view (620x620 μm/512x512 pixels) and deep penetration (750 μm beneath the dura).
We combined NIR-II illumination at ~1.7 μm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with high spatial resolution in adult mouse brain in vivo, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added as an additional channel to any laser-scanning microscope with low-cost sources and detectors, such as continuous-wave (CW) diode lasers and InGaAs photodiodes. The technique is label-free, simple and requires low illumination power, potentially creating new opportunities for deep tissue imaging in various biological and clinical applications.
We demonstrate three-photon microscopy (3PM) of mouse cerebellum at 1 mm depth by imaging both blood vessels and neurons. We compared 3PM and 2PM in the mouse cerebellum for imaging green (using excitation sources at 1300 nm and 920 nm, respectively) and red fluorescence (using excitation sources at 1680 nm and 1064 nm, respectively). 3PM enabled deeper imaging than 2PM because the use of longer excitation wavelength reduces the scattering in biological tissue and the higher order nonlinear excitation provides better 3D localization. To illustrate these two advantages quantitatively, we measured the signal decay as well as the signal-to-background ratio (SBR) as a function of depth. We performed 2-photon imaging from the brain surface all the way down to the area where the SBR reaches ~ 1, while at the same depth, 3PM still has SBR above 30. The segmented decay curve shows that the mouse cerebellum has different effective attenuation lengths at different depths, indicating heterogeneous tissue property for this brain region. We compared the third harmonic generation (THG) signal, which is used to visualize myelinated fibers, with the decay curve. We found that the regions with shorter effective attenuation lengths correspond to the regions with more fibers. Our results indicate that the widespread, non-uniformly distributed myelinated fibers adds heterogeneity to mouse cerebellum, which poses additional challenges in deep imaging of this brain region.
The attenuation of excitation power reaching the focus is the main issue that limits the depth penetration of highresolution imaging of biological tissue. The attenuation is caused by a combination of tissue scattering and absorption. Theoretical model of the effective attenuation length for in vivo mouse brain imaging has been built based on the data of the absorption of water and blood and the Mie scattering of a tissue-like phantom. Such a theoretical model has been corroborated at a number of excitation wavelengths, such as 800 nm, 1300 nm , and 1700 nm ; however, the attenuation caused by absorption is negligible when compared to tissue scattering at all these wavelength windows. Here we performed in vivo three-photon imaging of Texas Red-stained vasculature in the same mouse brain with different excitation wavelengths, 1700 nm, 1550 nm, 1500 nm and 1450 nm. In particular, our studies include the wavelength regime where strong water absorption is present (i.e., 1450 nm), and the attenuation by water absorption is predicted to be the dominant contribution in the excitation attenuation. Based on the experimental results, we found that the effective attenuation length at 1450 nm is significantly shorter than those at 1700 nm and 1300 nm. Our results confirm that the theoretical model based on tissue scattering and water absorption is accurate in predicting the effective attenuation lengths for in vivo imaging. The optimum excitation wavelength windows for in vivo mouse brain imaging are at 1300 nm and 1700 nm.
A designed multilayered metamaterial cavity formed by the metallo-dielectric multilayer structure (MDMS) and a nano
Aluminum layer coated substrate is exploited to achieve the sub-20 nm patterns feature sizes at the wavelength of 248
nm with p-polarization. The filtering and SPP cavity resonance coupling provided by this MDMS cavity regime enable
the SPP interference patterns with high uniformity and intensity output in the photoresist (PR) layer. Furthermore,
compared with the conventional grating metal waveguide structure, this lithography system demonstrates the better
stability of patterns period against the cavity thickness variation. The enhancement and the longitudinal extension of SPP
localized field offered by the proposed cavity scheme will provide a potential way to obtain the lithography patterns with
improved depth, contrast and perpendicularity.
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