SHG imaging of rat tail tendons ex vivo
was performed using the all-fiber-optic endomicroscopy system. Microscopically, the main constituent of tendon is collagen which comprises 80% dry weight of tendon [15
]. The tendon collagen fibers are mostly type I collagen, which is the most efficient SHG source in tissue due to the highly noncentrosymmetric structure [16
]. In this study, tendons were extracted from 5 fresh rat tails and kept in phosphate buffered saline (PBS) during imaging to prevent potential tissue dehydration. All imaging was conducted within two hours after the dissection of the rats. show representative real-time (at a frame rate of 3.3-frames/sec) and 10 frames averaged backward SHG images, respectively. The SHG signals were detected by a band-pass filter with a pass-band of 400-420 nm. As displayed by the SHG images, fine tendon structures (i.e. collagen fiber bundles) are clearly identifiable. The collagen fiber bundles are closely packed with a diameter of ~2.8 μm which is consistent with the fiber bundle size (~0.4 - 4.8 μm) reported in literature [18
]. The 3D animation in shows the layered intrinsic SHG images of rat tail tendon with an axial step of 5 μm controlled by a precision translation stage. Each section represented the average of 10 frames. As shown in the animation, different collagen bundles can be observed at different depths. Moreover, all the collagen fiber bundles at different depths are parallel. Overall, these preliminary ex vivo
imaging results are consistent with the general structures of tendon. It has been shown that modification of the collagen matrix in biological tissues is associated with various physiologic/pathological processes, such as wound healing, diabetes and cancer [19
]. Therefore, this compact all-fiber-optic SHG endomicroscopy system can be potentially used for real-time assessment of collagen fiber network morphology under various clinically relevant conditions for diagnosis and treatment monitoring.
Fig. 5 Representative intrinsic SHG images of rat tail tendon: (a) real-time image (3.3 frames/second) and (b) 10 frames averaged image. (c) animation of layered intrinsic SHG images as a function of depth. The SHG signals are solely attributed to type I collagen. (more ...)
Two-photon fluorescence (TPF) imaging of epithelial tissues was also performed using the scanning fiber-optic endomicroscopy system. and 7(b)
show the typical 10 frames averaged images of a formalin fixed pig corneal limbus (from the tissue surface) and corneal stroma (100 μm below the cornea surface), respectively. The tissue was stained after fixation with 1% acridine orange (AO) in PBS, the dye commonly used in single and two-photon fluorescence imaging for enhancing the nuclei contrast [21
]. The single-photon excitation/emission maximum of AO when bound to DNA are at 487/520 nm and the two-photon fluorescence spectrum with 780-900 nm excitation is similar to the single-photon fluorescence spectrum [23
]. The densely packed epithelial cells in the limbus (near tissue surface) and sparsely distributed keratocytes in the stroma (100 μm below the tissue surface) can be clearly identified from the images. In addition, the nuclei of epithelial cells and keratocytes exhibit different morphologies, which is consistent with the textbook histology [24
]. These images demonstrate that the system has sufficient resolution to resolve nuclei with a diameter of about 5 μm.
Fig. 6 Exogenous TPF images of pig cornea tissue stained with acridine orange. (a) corneal limbus and (b) corneal stroma. The densely packed epithelial cells in the limbus and sparsely distributed keratocytes in the stroma can be clearly identified from the (more ...)
To further demonstrate the potential of the endomicroscopy system for depth-resolved imaging of internal organs, 3D TPF images of fresh rat oral tissues stained with AO were acquired by axially translating the 2D-scanning endoscope with a precision translation stage. show representative images of the oral tissue at depths of 10 and 50 μm below the tissue surface, respectively. The movie in illustrates a series of en face
TPF images of the oral tissue as a function of depth. The depth separation between two adjacent images was 5 μm and the total imaging depth was 120 μm. The epithelial cell nuclei can be identified by the endomicroscopic TPF imaging. As seen from the depth-resolved images, the nucleus density increases from the superficial layer to the basal layer, which is consistent with the well-known histology of stratified squamous epithelium [24
]. Considering neoplastic oral mucosa exhibit depth-dependent changes in nuclei packing density and pleomorphism [25
], these ex vivo
two-photon imaging results strongly suggest a potential role of this system for neoplasia detection by real-time assessment of epithelial structures with cellular and subcellular resolution. Higher resolution to resolve more detailed intracellular structures is also essential and may be achieved by equipping the endomicroscope with a customized lens assembly with a higher NA.
Fig. 7 Typical depth-resolved TPF images of rat oral tissue stained with acridine orange: (a) at the depth of 10 μm; (b) at the depth of 50 μm; and (c) movie of stacked 3D images. The depth-resolved TPF images reveal that the nucleus density (more ...)