Microscopía multifotónica del ojo humano en vivodesarrollo y optimización de un dispositivo compacto para uso clínico

Abstract

In the past decades, multiphoton microscopy has become a very useful tool for the visualization of biological tissues. In particular, second harmonic generation (SHG) microscopy has an important advantage over conventional microscopy methods, due to its ability to observe tissues formed by type I collagen, such as the sclera and the cornea, without stanning and/or fixation procedures. These ocular structures are of interest for this PhD Thesis. For this purpose, we present a prototype of a compact multiphoton microscope adapted for the examination of the external segment of the living human eye. This new system requires a calibration of the acquisition parameters, aiming at a compromise between image quality and safety for the biological tissues under study, to record optimal images using reduced exposure times. The evaluation of the quality of images, recorded with different acquisition parameters, has confirmed that it is possible to achieve an optimal recording with exposure times below 1 s. Once the optimal recording conditions have been evaluated, it is necessary to know if the experimental system is suitable for in vivo imaging, taking into account the laser source parameters, the exposure time and the visual angle, following the ANSI Z136.1 standards. Therefore, a detailed analysis of the Maximum Permissible Exposure (MPE) calculation for the experimental system developed is included herein. This analysis concludes that the multiphoton microscope developed in this work is safe for the proposed experimental conditions. After the safety analysis, we proceeded to record images in live conditions of healthy volunteer patients, keeping the experimental conditions well below the calculated MPE. As a result, images of the cornea, sclera and sclero-corneal limbus were recorded with sufficient resolution to detect individual fibers and cells, comparable to ex-vivo samples, with high repeatability. In some cases, involuntary eye movements can cause image degradation, hence the need to reduce the exposure time, and, as in all biological samples, when images are obtained deeper in the eye, phenomena such as scattering occur, which also reduce the quality of the images. Due to this, different methods of image improvement are proposed. We have reported a marginal blind deconvolution method. This allows restoring images when little information is available from the experimental system, and unsupervised. This procedure has demonstrated to increase the quality of the deconvoluted images, as well as in their resolution, compared to the original images. An alternative enhancement method has been the use of radially polarized light, by means of an S-waveplate. The images acquired with radially polarized light have been compared with images recorded, quasi-simultaneously, with circularly polarized light. The increase in quality of images acquired with radial light is remarkable, especially noticeable with depth, where images are more degraded due to aberrations and scattering. Finally, we have compared images recorded with a sub-10fs laser, with reduced dimensions and greater flexibility for the implementation of a prototype multiphoton microscope in the clinic, and a typical Ti:Sapphire laser of more than 120 fs, of large dimensions and high cost. The results show that the sub-10fs laser is highly affected due to the action of chromatic dispersion, being necessary to increase the laser output power to obtain images of similar qualities to those acquired with the Ti:Sapphire laser. The enhancement methods applied in this Thesis allowed enhanced multiphoton images that provide information on the structures of the cornea and sclera, while not compromising the integrity of the tissues, since they remain several orders of magnitude below the maximum exposure levels allowed for the human eye.