Purchase this article with an account.
H. Nakamura, Y. Zhao, Y. Liu, B. Y. J. T. Yue, R. J. Gordon; Two-Photon Microscopy for Real Time Imaging of the Aqueous Outflow Pathway. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5559.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the structure and function of the aqueous outflow pathway of the eye, a two-photon fluorescence microscope was built, designed specifically for real time imaging of the eye.
A prototype two-photon microscope designed for imaging eye tissue was built. Briefly, 35 femtosecond 800 nm laser pulses, generated from a commercial mode-locked Ti:Sapphire oscillator (Tsunami, Spectra Physics), were steered by a 45o dichroic mirror and focused onto the sample surface by a 40X, 0.66 NA objective. The fluorescence induced by the focused laser pulses was collected by the same objective and detected by a photomultiplier tube (PMT), after passing through the dichroic mirror and a short-pass filter. The current from the PMT was fed into a computer equipped with a data acquisition card. To acquire images, a raster scan of the laser focus within the xy focal plane was produced using a 2D galvanometer scanner, which was driven by a two-channel arbitrary waveform generator. A homemade computer interface was developed for laser scanning control, signal detection, and display of the two-photon images. The field of view and resolution of the microscope were determined using artificial targets. To investigate the phototoxicity of the laser radiation, cultured human trabecular meshwork (TM) cells were ablated by the laser under conditions identical to those used during microscopic examinations. An MTT assay of the TM cells was performed to determine the phototoxicity of the laser as well as the cytotoxicity of various fluorescent cell labeling reagents including QtrackerTM. In addition, images the TM tissue were acquired with the two-photon microscope.
The resolution of the microscope was determined to be 0.63 µm in the lateral (xy) plane and 3.8 µm in the z-direction. The observable area was 150 x 150 µm, the working distance was 0.49 mm, and the scanning time was 20 sec/frame. No laser phototoxicity was detected by the MTT assay. Among the reagents tested, QtrackerTM did not show any cytotoxicity. TM beams as well as cells in the human specimens were visualized and images were captured by the prototype of two-photon microscope.
A prototype of a two-photon microscopy system designed specifically for eyes was built and characterized. Real time observations to investigate the aqueous outflow pathway of the eye are feasible using this instrument.
This PDF is available to Subscribers Only