Nonlinear Endomicroscopy

for visualization of histology in situ and in real time by structural, molecular and biochemical/metabolic imaging of biological tissues at cellular and subcellular level (0.5-2 μm resolution, 0.2-0.5 mm imaging depth).

Nonlinear Endomicroscopy, including two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy, is a powerful imaging technique for 3D structural and functional imaging of biological tissues at true histopathology resolution. Our goal is to miniaturize a bench-top TPF/SHG microscopy system to a flexible fiber-optic endomicroscope (2-3 mm in diameter) to enable a variety in vivo and clinical applications. In 2006, we reported the first all-fiber-optic fully integrated scanning endomicroscope for two-photon fluorescence imaging. The probe had a small diameter (2.4 mm) and an ultra light weight (~0.5 g).

Figure 1. Real-time visualization of histology: from an scanning laser two-photon fluorescence and SHG microscope to a flexible fiber-optic endomicroscope and representative TPF and SHG endomicroscopy images. (Click to enlarge and view detailed caption)

Rapid spiral beam scanning with a fiber-optic scanner Illustration of dispersion compensation

Animation 1. Animations of fiber-optic beam scanning and dispersion compensation. (Click to play and view detailed caption)

Figure 2. Schematics and Photos of a fiber-optic scanning endomicroscope for two-photon fluorescence and SHG imaging. (Click to enlarge and view detailed caption)

There are many technical challenges associated with this research endeavor. The major challenges include single-mode delivery of fs excitation pulses, efficient collection of TPF/SHG signals, an ultra-compact fast beam scanning mechanism, and high-quality miniature optics to handle the large separation between the near-infrared excitation and visible TPF/SHG wavelengths. Our research focuses on addressing these critical technological challenges by developing/exploring new fiber-optics, miniature imaging optics, and MEMS technologies. In addition, potential applications of the fiber-optic endomicroscopy technology for in vivo imaging are also being carried out to explore new or true functional/metabolic/biochemical information/mechanisms that are otherwise difficult or impossible to obtain.

Figure 3. SHG endomicroscopy images of mouse cervical tissues with the corresponding histology slides. See more details in Y. Y. Zhang, et. al., PNAS 109 (2012) (Click to enlarge and view detailed caption)

One potential clinical application of this technology is for pre-term birth prediction as described in Preterm Risk Assessment.

Pig cornea tissue Rat oral tissue

Animation 2. 3D animations of multiphton endomicroscopy images. (Click to play and view detailed caption)




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