Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

We will empower the study of biological processes in living animals through the real-time visualization, implanting a miniaturized chip, manufactured by 3D-laser printing of a biocompatible material.

The main challenge is fine-tuning the fabrication parameters, such as power and speed, considering different writing conditions, meanwhile, microstructure in both surfaces of the same subset with precision and consistency. Accurate result is establishing a versatile protocol for fabricating an innovative, implantable, optical imaging tool, directly coupling large micro lenses to 3D microstructure target region for various biological applications.

Now that the fabrication protocol has been optimized, we are working on the implantation and demonstration of the imaging capabilities of the chip. For example, for in vivo myo-material testing.

[AI instructor] To begin, switch on the femtosecond near infrared laser source. Align the laser beam's optical path until it reaches the microscope objective through a series of optics and mirrors mounted on kinematic mirror mounts. Iteratively rotate the mirrors to center the beam within near infrared alignment. Pinholes direct the laser beam perpendicularly to the sample holder by aligning it using back reflection centering. To mount the sample on the sample holder, use tape to fix the double-dropped glass cover slip on the sample holder with the second deposited drop facing downward. Then mount the sample holder on the translation stages, manually mount the sample holder, then mount the long working distance microscope objective on the dedicated support at the end of the optical path, close to the sample, and center the sample with the objective. Set the laser power to the minimum value, approximately five milliwatts, sufficient to visualize the beam reflection on the CCD camera software. Focus the laser beam on the upper surface of the first resist drop. Follow the curved profile of the drop to locate the sample edges along the x and y directions. Set the center of the drop as an absolute zero reference using the software. Focus the laser beam on the interface between the upper surface of the glass cover slip and the base of the first drop of photoresist at the center of the sample. Set this as zero reference on the z axis. Move to the edge position in the negative x axis direction for approximately 3.5 millimeters for a 12-millimeter cover slip and focus on the same interface. Set this as the absolute zero reference along the z direction. Repeat the same for the positive x axis direction for approximately 3.5 millimeters and focus on the same interface. Then tilt the sample to correct for deviations in the z direction between the negative and positive x axes. Perform the same procedure as demonstrated previously along the x axis for the y axis. Once balanced on both the x and y axes, return to the central position and focus on the interface between the glass and the resist. Set the new z value of the focus as zero reference on the z axis. Switch on the red LED illumination system for real-time monitoring of the polymerization process. With the laser off, move the objective along the z direction below the glass cover slip to locate the second interface between the bottom surface of the glass and the base of the lower drop of resist. Increase the laser power to 100 milliwatts to initiate two-photon polymerization. Tune the focal position by increasing z until a simple reference structure is polymerized. Set this initial focal position as zero reference along the z axis. Set polymerization powers between 100 and 200 milliwatts and run the machine code as a computer numerical control program for the translational stages to fabricate the desired three-dimensional structure. Next, move along the z axis to return to the first interface between the upper glass surface and the upper drop of photoresist. Polymerize a simple reference structure to locate the interface. Set the first line of polymerization as zero reference along the z axis. Adjust the polymerization power between 15 and 20 milliwatts and run the program guiding the translational stage movements. With the laser off, disable the x, y, and z translational axes and remove the sample holder from the experimental fabrication setup. Heal off the sticky tape and detach the sample from the holder. After sample development place the glass cover slip on a sample holder suspended from the ground plane laying the sample with the micro lenses facing downward. Position the sample under the UV source oriented perpendicularly with respect to the surface of the glass cover slip. Expose the sample to UV radiation. Set at 300 milliwatts for 120 seconds. Tilt the UV source to plus and minus 45 degrees with respect to the normal position of the sample plane and repeat the exposure procedure. Place the glass sample on the holder at a 45-degree angle with respect to the orientation of the SEM camera. Repeat the acquisition process for both surfaces of the glass cover slip to collect three dimensional SEM images of the micro scaffolds and micro lenses. The presented procedure allows for the polymerization of 3D microstructures of both surfaces of the same device, ensuring excellent resolution and stability. In vitro imaging showed successful growth of cells inside the micro scaffold, imaged through the micro lenses, representing an example of a final application of the proposed device.