Abstract
Purpose: :
To determine the safety of examining live human cornea in vivo with the 2-photon ophthalmoscope by initial exposure of human eye bank corneal buttons.
Methods: :
Specular microscopy and vital staining (Trypan Blue/Alizarin Red) were used to determine cell loss on corneal tissue after exposure to a two-photon ophthalmoscope within laser safety limits.Laser Safety Calculation:According to Delori et al. (2007) and ANSI laser safety guidelines (Maximum permissible exposures for ocular safety, 2000), with emphasis on ophthalmic devices, with our system settings (central wavelength =780 nm, pulse width=150 fs, repetition rate =78 MHz, numerical Aperture= 0.8, irradiated area= 1536µm * 1536µm =0.0236cm2), the Maximum Permissible corneal irradiance is 94.4mW for long radiation (t>10s) and for shorter radiation the power limit for 6 frames (1.2s) is 515mW.Specular Microscopy and Vital Staining:Seven normal corneal buttons were prepared by the San Diego Eye Bank then placed in artificial anterior chambers. Five corneas were exposed on stage under the 2-photon scope (Exposed) and 2 buttons were placed on a table at room temperature (Controls). Laser power was set at a high level for Exposed buttons, 170mW. Exposure time ranged from 10 seconds to 1 hour and depth of tissue ranged from 0 (corneal surface) to 300 microns. Cell loss was determined by specular microcopy changes as well as final Trypan Blue/Alizarin Red staining using the Adobe Photoshop Method (Saad, et al., 2008).
Results: :
2-photon laser Exposed corneas had cell loss range between 0% and 30.8%, depending on the technique used (specular microscopy vs vital staining, respectively), while Controls had loss of 4.26% to 30.0 %, respectively. Maximum loss between both groups was comparable. Vital staining in both groups was minimal and occurred mostly on the periphery of the cut buttons, while sparing the central endothelium.
Conclusions: :
The results of these findings indicate that the 170 mW 2-photon Exposed buttons were comparable to the Control buttons, demonstrating minimal endothelial cell loss, even up to 1 hour exposure and at 300 microns depth. Tested on more observations, this prototype may provide the possibility to non-invasively examine eyes in vivo, thereby allowing earlier detection and diagnosis of corneal diseases.
Keywords: cornea: endothelium • laser • imaging/image analysis: clinical