Although fiber-optical two-photon endoscopy has been recognized as a potential high-resolution diagnostic and therapeutic procedure in vivo, its resolution is limited by the optical diffraction nature to a few micrometers due to the low numerical aperture of an endoscopic objective. On the other hand, stimulated emission depletion (STED) achieved by a circularly-polarized vortex beam has been used to break the diffraction-limited resolution barrier in a bulky microscope. It has been a challenge to apply the STED principle to a fiber-optical two-photon endoscope as a circular polarization state cannot be maintained due to the birefringence of a fiber. Here, we demonstrate the first fiber-optical STED two-photon endoscope using an azimuthally-polarized beam directly generated from a double-clad fiber. As such, the diffraction-limited resolution barrier of fiber-optical two-photon endoscopy can be broken by a factor of three. Our new accomplishment has paved a robust way for high-resolution in vivo biomedical studies.
The technique has potential uses for brain imaging, early cancer detection and minimally invasive surgical procedures as well as the development of new tests for drugs and other treatments.
Experimental results have demonstrated that the diffraction-limited resolution barrier can be broken by a factor of 3. Consequently, the highest image resolution of 310 nm is achieved for the endoscopic probe of NA = 0.35, which should correspond to the image resolution of 75 nm if the NA was 1.2.
Further increasing the image resolution in our system requires the higher depletion power, which may not be realistic and could lead to the damage to a sample when the depletion power is higher than 100 mW. However, a factor-of-three increase in the image resolution is an optimized result for the given microsphere sample used in this paper. To confirm this point, we theoretically reveal the fiber-optical STED two-photon fluorescence microscope with NA = 1.2 can result in the image resolution of 75 nm for the depletion power of 100 mW due to the high peak intensity of the depletion beam, revealing an increase of the image resolution by a factor of three. Of course, if fast fluorophores with a steep depletion curve or a large depletion rate (ζ) were used, our fiber-optical STED two-photon fluorescence endoscope could result in the image resolution better than 100 nm. Alternatively, a fast scanning mechanism is capable of enhancing the depletion efficiency by avoiding the re-excitation of a fluorophore.
It should be noted that improving the axial resolution with the STED principle in the DCF endoscope system might also be possible. One of the ways is to use the superposition of a radial polarization beam with an azimuthal polarization beam in combination with a concentric π phase-shift wave plate, which can produce a 3D confinement in the focus and can be adopted for improving axial resolution35. The approach can be implemented in the DCF, although this process might increase the complexity of the illumination optical setup.
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