Using short-wave infrared wavelength advantages, we demonstrate one-photon fluorescence confocal microscopy of adult mouse brains with penetration depths up to 1.7mm. This is achieved by labeling quantum dots with 1300 nm excitation and 1700 nm emission and detecting them with a single-photon superconducting nanowire detector.
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 demonstrate a multi-color background-free coherent anti-Stokes Raman scattering (CARS) imaging system, using a low-cost, all-fiber, energetic, multi-wavelength time-lens source. The time-lens source generates picosecond pulse trains at three different wavelengths. The first is 1064.3 nm, the second is tunable between 1052 nm and 1055 nm, and the third is tunable between 1040 nm and 1050 nm. When the time-lens source is synchronized with a mode-locked Ti:Sa laser, two of the three wavelengths are used to detect different Raman frequencies for two-color on-resonance imaging, whereas the third wavelength is used to obtain the off-resonance image for nonresonant background subtraction. Mixed poly(methyl methacrylate) (PMMA) and polystyrene (PS) beads are used to demonstrate two-color background-free CARS imaging. Synchronization of the multi-wavelength time-lens source with a microscope enables pixel-to-pixel wavelength-switching. Simultaneous two-color CARS imaging of CH2 and CH3 stretching vibration modes with realtime background subtraction is demonstrated in ex vivo mouse tissue.
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.
We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering (CARS) imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm (~ 400 mW for each wavelength). When synchronized to a modelocked Ti:Sa laser, the two wavelengths are used to obtain on- and off-resonance CARS images. Real-time subtraction of the nonresonant background in the CARS image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free CARS imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.