This content will become publicly available on May 24, 2023
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- Frontiers in Neuroscience
- Sponsoring Org:
- National Science Foundation
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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 [1, 2]. 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 moremore »
The light attenuation in biological tissues, caused by scattering and absorption, is a main issue in deep imaging. While the signal from the focal volume in multiphoton imaging is mostly generated by ballistic photons, both ballistic and scattered fluorescence photons contribute to the detected signal. The impact of emission wavelengths on deep imaging has not been investigated experimentally before. Here we perform a systematic comparison of the fluorescence attenuation in tissue at the emission wavelengths of 520 nm, 615 nm and 711 nm in three-photon imaging of mouse brain vasculature in vivo. Our results show that the impact of the emission wavelengths on multiphoton imaging depth is small compared to the excitation wavelengths.
Multiphoton fluorescence microscopy is a powerful technique for deep-tissue observation of living cells. In particular, three-photon microscopy is highly beneficial for deep-tissue imaging because of the long excitation wavelength and the high nonlinear confinement in living tissues. Because of the large spectral separation of fluorophores of different color, multicolor three-photon imaging typically requires multiple excitation wavelengths. Here, we report a new three-photon excitation scheme: excitation to a higher-energy electronic excited state instead of the conventional excitation to the lowest-energy excited state, enabling multicolor three-photon fluorescence imaging with deep-tissue penetration in the living mouse brain using single-wavelength excitation. We further demonstrate that our excitation method results in ≥10-fold signal enhancement for some of the common red fluorescent molecules. The multicolor imaging capability and the possibility of enhanced three-photon excitation cross section will open new opportunities for life science applications of three-photon microscopy.
Multiphoton fluorescence microscopy enables deep
in vivoimaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedented in vivoimaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.
The mouse subventricular zone (SVZ) produces neurons throughout life. It is useful for mechanism discovery and is relevant for regeneration. However, the SVZ is deep, significantly restricting live imaging since current methods do not extend beyond a few hundred microns. We developed and adapted three-photon microscopy (3PM) for non-invasive deep brain imaging in live mice, but its utility in imaging the SVZ niche was unknown. Here, with fluorescent dyes and genetic labeling, we show successful 3PM imaging in the whole SVZ, extending to a maximum depth of 1.5 mm ventral to the dura mater. 3PM imaging distinguished multiple SVZ cell types in postnatal and juvenile mice. We also detected fine processes on neural stem cells interacting with the vasculature. Previous live imaging removed overlying cortical tissue or lowered lenses into the brain, which could cause inflammation and alter neurogenesis. We found that neither astrocytes nor microglia become activated in the SVZ, suggesting 3PM does not induce major damage in the niche. Thus, we show for the first time 3PM imaging of the SVZ in live mice. This strategy could be useful for intravital visualization of cell dynamics, molecular, and pathological perturbation and regenerative events.