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.
A miniaturized scanning mechanism is a crucial component in the creation of endoscopes for microscopic imaging.
Several groups have developed resonant scanners (e.g., spiral or Lissajous scan pattern), but these suffer from limitations
in non-uniform spatial coverage and sampling time, in comparison to a raster scanner. Additionally, a resonant scanner
lacks the ability to perform line-scan imaging, a crucial capability in measuring a variety of fast, dynamic physiological
phenomena (e.g., blood flow, molecular diffusion, etc.). However, current miniaturized raster scanners are limited in
terms of their physical dimensions and scan speed. We demonstrate a novel hybrid resonant/non-resonant miniaturized
raster scanner, fabricated by mounting a double clad fiber onto two perpendicularly oriented piezo bimorphs. The fiber
scanner has a total length of 2.6cm, a width/thickness [less than or equal to] 1mm, achieves [greater than or equal to] fiber tip deflection for both the resonant
and non-resonant axes, and allows for imaging at approximately 4 frames per second (512 lines per frame). An
essentially uniform spatial coverage and sampling time can be achieved by utilizing the middle portion (e.g., middle 500
μm) of the resonant scanning range. The small size allows for the fiber scanner to be easily packaged along with
miniaturized lenses to form an endoscope for microscopic imaging. We bonded a stiffening fiber alongside the vibrating
fiber to break its cylindrical symmetry. Thus, only one vibration mode is excited, generating a purely linear spatial
motion. In order to demonstrate the fiber scanner's imaging capabilities we have taken transmission and fluorescence
images, in which the double clad fiber's inner clad is used for fluorescence collection.
As a result of the large difference between scattering mean free paths and absorption lengths in brain tissue, scattering
dominates over absorption by water and intrinsic molecules in determining the attenuation factor for wavelengths
between 350 nm and 1300 nm. We propose using longer wavelengths for two-photon excitation, specifically the 1300-nm region, in order to reduce the effect of scattering and thereby increase imaging depth. We present two photon
fluorescence microscopy images of cortical vasculature in in vivo mouse brain beyond 1 mm. We also explore the
capabilities of the 1300-nm excitation for third harmonic generation microscopy of red blood cells in in vivo mouse
We quantitatively compared the maximal two-photon fluorescence microscopy imaging depth achieved with 775 nm
excitation to that achieved with 1300 nm excitation through ex-vivo TPM of blood vessels in the mouse brain. We
achieved high contrast imaging of labeled blood vessels at approximately twice the depth at 1300 nm excitation as at 775
nm excitation. We also measured the two-photon excitation
cross-sections of several commercially available
fluorophores at 1220-1320 nm. We found that some of these fluorophores reveal comparable if not better cross-section
values than those of the widely used dyes excited by shorter wavelength light.