Two-photon (2P) excitation microscopy has opened up new avenues for biomedical investigations in living tissues [1
]. Superior properties of 2P microscopy, including intrinsic sectioning ability that obviates the need for a confocal pinhole, increased penetration depth in light-scattering media through the use of a longer wavelength (red or infrared) excitation laser, and reduced out-of-focus photobleaching, make it an indispensable tool for high-resolution imaging of structures in thick tissues and in live animals. Although many applications have been reported using bulky, bench-top 2P microscopes to image outer tissue layers [3
], there is a clear need for a miniaturized and flexible 2P endoscope that would allow imaging of inner organs [4
]. Driven by this need, 2P endoscopy has advanced rapidly, benefiting from improvements in optical fibers and micro-optical and micro-mechanical components [6
Many advances in 2P endoscopy have been reported in the last decade [9
]. Most endoscope designs involve three key components: micro-optics used for imaging, optical fibers used for efficient laser delivery and signal collection, and miniaturized scanning devices. The micro-sized imaging optics usually consist of either gradient-index (GRIN) lenses or compound lenses [15
]. Several types of optical fibers have been used for construction of endoscopes, including single mode fibers [16
], conventional double cladding fibers [13
], photonic crystal fibers (PCFs) [12
], and two-fiber systems consisting of one hollow core PCF for laser delivery and another multimode fiber for fluorescence signal collection [14
]. The two prevailing choices for the scanning unit are microelectromechanical systems (MEMS) based on small scanning mirrors [12
] and mechanically scanned fiber cantilevers driven by a piezoelectric transducer (PZT) [13
]. Some endoscopes avoid introducing a compact scanner by using long cylindrical GRIN relay lenses [6
] or a coherent fiber bundle [9
] to project the laser focus. With these designs, the laser focus is scanned by bulky components located outside of the endoscope, at the cost of flexibility and resolution. Among the various designs, light-weight and catheter-like 2P endoscopes employing PZT-driven fiber cantilever scanners and GRIN lenses have been demonstrated to be advantageous over other endoscopy technologies in terms of size, weight, and flexibility [4
]. Despite these advances, few clinical or biological applications of 2P endoscopes for tissue imaging have been reported [14
], largely because of the challenges of efficient fluorescence signal collection [15
]. Especially for applications involving intrinsically weak autofluorescence signals, as, for example, from NADH [23
], the signal-to-noise level of fiber-based endoscopy is still far from satisfactory.
Here we report the design and construction of a miniaturized, fiber-based 2P endoscope, intended for real-time imaging of the aqueous outflow pathway, including the trabecular meshwork (TM) in the eye. The TM is the primary site for regulation of the normal bulk flow of the aqueous humor [24
]. Abnormalities in the structure and function of the aqueous outflow pathway, including the TM, are considered to be one of the major reasons for elevated intraocular pressure in primary angle open glaucoma [24
]. A real-time imaging tool is needed in this field because conventional histological methods are plagued with artifacts introduced during the tissue-fixing procedure that have led to confusion and debate in the ophthalmological literature [26
]. Recently, 2P microscopy has been shown to be a promising tool for investigating the structure and function of the aqueous outflow pathway [28
]. Using this approach to image living eye tissues with commercially available 2P microscopes is impractical, however, because such instruments require the subject to be immobilized and affixed to a glass slide mounted on the microscope stage [30
]. The flexibility of a compact catheter-type 2P probe promises to overcome these obstacles by allowing ab interno
imaging of the eye.
In order to explore the feasibility of employing 2P endoscopy for ex vivo
study of the aqueous outflow pathway using an anterior segment perfusion culture system of the eye, we constructed a light-weight, catheter-like microscope and demonstrated its capability for imaging human TM. The miniaturized microscope consists of a PZT-driven resonant fiber scanner, a silica double cladding fiber, and three doublet achromatic lenses. Different lens configuration and fiber options were investigated during the construction of the instrument in order to obtain good spatial resolution and high fluorescence collection efficiency. Doublet chromatic lenses and a double cladding fiber were chosen for the instrument primarily to enhance its fluorescence signal collection efficiency, as compared with that obtained with other 2P microscopes [13
]. In particular, three achromatic doublet lenses were used instead of GRIN lenses to reduce achromatic aberration [31
]. A silica double cladding fiber with a high numerical aperture (NA) in the inner cladding was selected to optimize the balance among the signal collection efficiency, imaging resolution, and autofluorescence background inside the fiber. Simple methods are demonstrated in this paper to reduce image distortions that are inherent with resonant fiber scanner imaging modalities. Optimization of the signal to noise level enabled us to obtain good 2P images of human TM tissue, thereby laying the foundation for further ex vivo
or in vivo
investigation of the aqueous humor outflow pathway.
This paper is organized as follows. Details of the instrument design are presented in the following section. The performance of its various components and the motivation behind their design are discussed in Section 3. Section 4 presents the application of the endoscope to imaging of the TM tissue in vitro. Suggestions for future improvements and a summary are presented in the final two sections.