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Hiroshi Nakamura, Youbo Zhao, Robert J. Gordon; Two-Photon Microscopy and Endoscopy for Real Time Imaging of the Aqueous Outflow Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1347.
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© ARVO (1962-2015); The Authors (2016-present)
Ex vivo imaging of the aqueous outflow pathway ab externo was performed using a two-photon (2P) microscope, which we designed for real time imaging of the eye (ARVO 2010 E-Abstract 5559). In addition, we built and tested a 2P endoscope suitable for imaging ab interno.
Human eye-bank eyes were obtained from the Illinois Eye Bank. Within 48 hours after death, the anterior segment was dissected and clamped in a perfusion culture device. Phenol red-free DMEM was used to perfuse the anterior segment. The perfused pressure was controlled by the height of the solution reservoir relative to that of the eye. For imaging ab externo, the inner wall of Schlemm’s canal was surgically bared by a sinusotomy procedure. Living cells were stained with LavaCell® fluorescence dye both in perfused DMEM and in PBS, which was applied to the observed site ab externo at concentrations of 5 and 20 uM, respectively. The inner wall was observed by 2P microscopy, with lateral and axial resolutions of 0.49 um and 2.7 um, respectively. The main constituents of the 2P endoscope are: (i) a double-clad fiber, which delivers the laser pulses through its core and collects the fluorescence in its inner cladding, (ii) a miniature piezoelectric tube used to drive the distal tip of the fiber, scanning in a spiral pattern, and (iii) an assembly of three achromatic lenses for focusing the laser and collecting the 2P fluorescence. All components are enclosed in a 12-gauge hypodermic tubing with an outer diameter of 2.8 mm and a length of 31 mm.
Using the 2P microscope, various sizes of giant vacuoles (GVs, 2 to 24 um diameter, 25 cmH2O of perfusion pressure) were clearly detected near the surface of the inner wall, while the structure of the trabecular meshwork (TM) was observed in deeper sections. The 2P endoscope has an imaging speed of 1.1 frames/second and a field of view of 150 um in diameter, with lateral and axial resolutions as 1.5 um and 9.2 um, respectively. TM beams as well as cells in the fixed specimens were visualized with this instrument.
Real-time imaging with a 2P microscope is a promising procedure for investigating structure and function of the aqueous humor outflow pathway ab externo. Imaging ab interno with a 2P endoprobe is also feasible, and further improvement of its resolution is anticipated.
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