Deep tissue in vivo two-photon fluorescence imaging of cortical vasculature in a mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.6 mm in mouse cortex is achieved in ...vivo, approximately reaching the fundamental depth limit in scattering tissue.
Two-photon fluorescence microscopy (2PM)
enables scientists in various fields including neuroscience
, embryology
, and oncology
to visualize
and
tissue morphology and physiology at a cellular level ...deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue
or the insertion of optical probes
. Here we demonstrate non-invasive, high resolution,
imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.
We compare the maximal two-photon fluorescence microscopy (TPM) imaging depth achieved with 775-nm excitation to that achieved with 1280-nm excitation through in vivo and ex vivo TPM of ...fluorescently-labeled blood vessels in mouse brain. We achieved high contrast imaging of blood vessels at approximately twice the depth with 1280-nm excitation as with 775-nm excitation. An imaging depth of 1 mm can be achieved in in vivo imaging of adult mouse brains at 1280 nm with approximately 1-nJ pulse energy at the sample surface. Blood flow speed measurements at a depth of 900 mum are performed.
We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens ...assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.1 frames/s (512 lines per frame) is achieved by simultaneously driving the DCF cantilever at its resonant frequency in one dimension and nonresonantly in the orthogonal axis. The implementation of a DCF into the scanner enables simultaneous delivery of the femtosecond pulsed 800-nm excitation source and epi-collection of the signal. Our device is able to achieve a field-of-view (FOVxy) of 110 μm by 110 μm with a highly uniform pixel dwell time. The lateral and axial resolutions for two-photon imaging are 0.8 and 10 μm, respectively. The endoscope’s imaging capabilities were demonstrated by imaging ex vivo mouse tissue through the collection of intrinsic fluorescence and second-harmonic signal without the need for staining. The results presented here indicate that our device can be applied in the future to perform minimally invasive in vivo optical biopsies for medical diagnostics.
Multiphoton microscopy (MPM) is widely used for optical sectioning deep in scattering tissue,
1-2. Phosphorescence lifetime imaging microscopy (PLIM) 3 is a powerful technique for obtaining ...biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching 4-5. Point-measurement PLIM 6 of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM 7, 8. However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here we experimentally demonstrate a parallel-excitation/parallel collection MPM-PLIM system that increases pixel rate by a factor of 100 compared with conventional configurations while simultaneously acquiring lifetime and intensity images at depth
. Full-frame three-dimensional in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging.
We present a technique for tunable dispersion compensation that is low cost, high speed, and has a large tuning range. By rotating a cylindrical lens at the Fourier plane of a folded 4f grating pair ...system, the group-velocity dispersion can be tuned over a range greater than 10(5) fs(2), sufficient for compensating the dispersion of several meters of optical fiber.
Deep tissue multiphoton imaging of mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.5-mm in mouse cortex is achieved in vivo, approximately reaching the fundamental ...depth limit in scattering tissue.
We demonstrate 67-nJ, 65-fs soliton pulse generation using a solid-core photonic crystal rod pumped by a compact fiber source, and its application to in vivo three-photon microscopy in mouse brain.
We demonstrate high-energy soliton pulse generation from a large mode area fiber, tunable from 1560 nm up to 1700 nm, pumped with a 10 MHz, 1.5-ps time lens source at 1544 nm.
We demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures (the external capsule (EC) and hippocampus) within an intact mouse brain using three-photon fluorescence ...microscopy at the new spectral window of 1700 nm.
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