Subproject 5 – Microendoscopy

Participants: Cícero Omegna (Kom-Lux) and Carlos Lenz Cesar (IFGW-Unicamp)

There is a large international effort in the area of microendoscopy with the use of photonic microscopies because the will allow in vivo studies. The idea behind microendoscopy is to make a laser beam emmited by an optical fiber, or a fiber cable, to execute a scanning and to use the same fiber to collect the optical signals generated in the sample. The great chalenges in high resolution microendoscopy are to scan the laser, to eliminate the pinhole, increase the numerical apperture of the excitation and emission light and, (if based on fluorescence) to label the cells. This project present prospects to circunvent each of these problems. To avoid the use of a pinhole, we will use non-linear optics, which are intrinsically confocal. SHG/THG and CARS do not rely on neither autofluorescence nor the use of fluorochromes, though naturally fluorecent molecules, such as tryptofan, indolamines (including ther dimers and trimers), fibrilins, NADH, elastin, lipofuscin and flavins. Of these, only the flavins could be excited with wavelenghts over 400 nm, meaning that excitation should be at the UV region, in which the attenuation of optical fibers are very high. However, this would not be a problem for multiphoton microscopies, because the excitation uses 2 or 3 photons from the infrared region for the which optical fibers are particularly transparent. If non-linear optics solve the need for a pinhole and UV excitation, it faces a new challenge. Optical fibers widen the light pulse period due to a phenomenon known as group velocity dispersion. Each pulse is formed by various components with different wavelengths. After group velocity dispersion, each of these components propagate at different velocities, widening the pulse period. A pulse that enters an optical fiber with 100 femtoseconds might leave it with tens of picoseconds, decreasing the efficiency of multiphoton lasers. Special photonic optical fibers are available which reduces to a large extent the group velocity dispersion. Other techniques exist to compensate this phenomenon and ensure the stability of pulse period leaving an optical fiber. The pourpose of this project is to use both mechanisms to solve this problem. Two chalenges remain, which are related to the characteristics of optical fibers. How to perform laser scanning and how to obtain high numerical appertures. Scanning is a challenge for all confocal microendoscopy techniques. The suggestion of a distal scanning, at the optical fiber end in contact with the sample are based on micromirror controlled by MEMS [Micro-Electro-Mechanical Systems] or on the rotation of micro-primas or special fibers. The need to transport electric or mechanical energy to the end of fibers tend to increase the dimension of the endoscope. So, we believe that proximal scanning, at the opposing end is more useful. For this, the challenge is to translate the scanning from one end of the fiber to the other. This is easily achieved by using a coherent cable of fibers, that might have more than one thousand fibers. This will be one of our strategies, even though realizing that lateral resolution, defined as the distance between optical fibers in a cable, will be low. Papers in the literature show that this strategy is possible with lateral resolution of 2 to 3 mm and axial resolution of 15 mm (J. Vasc. Res. 41: 400, 2004; Opt. Express 15: 4008, 2007). However, the best results were obtained with the use of (GRaded-INdex Fiber) optical fibers. In these fibers, the refraction index is higher in the center than in the periphery, making the rays to curve toward the center as in a lens. Using a fiber lentght so that each end correspond to conjugated optical planes, a scanning procedure at one end is completely transferred to the other. One micro-lens in one end of the fiber allow us to get thigh numerical apperture. Using two-photon microscopy and GRIN fibers, the German GRINTECH has built microendoscopes with numerical appertures as high as 0.85 and lateral resolution of 500 nm and axial resolution of 1400nm. The potential is to go up to numerical appertures as high as 1.4. The objectives of this projectare (1) Modify optical fiber cables in na endoscope including a GRIN fiber in the center and (2) use a confocal microscope scan head to scan the laser at the distal end of the GRIN fiber. So, the scanning will be transferred to the proximal end of the fiber in contact with the sample; (3) adapt the cable illumination system to operate conventionally, using lamp bulbs instead of lasers. Scanning will be done in the cable as a whole for the image acquisition with less resolution, or only in the central region, coincident with the GRN fiber, for the acquisition of high resolution images. We expect to develop na accessory for confocal microscopy that can be adapted to any commercial microscope and to show the possibility of produding a complete photonic-based microendoscope. This prototype will then be shared with members of this research team and KOM-LUX will study the best way to turn it into a commercial product.