Multiphoton microscopy (MPM) has become a preferred technique for intravital imaging deep in living tissues with subcellular detail, where resolution and working depths are typically optimized utilizing high numerical aperture, water-immersion objectives with long focusing distances. However, this approach requires the maintenance of water between the specimen and the objective lens, which can be challenging or impossible for many intravital preparations with complex tissues and spatial arrangements. We introduce the novel use of cohesive hyaluronan gel (HG) as an immersion medium that can be used in place of water within existing optical setups to enable multiphoton imaging with equivalent quality and far superior stability. We characterize and compare imaging performance, longevity, and feasibility of preparations in various configurations. This combination of HG with MPM is highly accessible and opens the doors to new intravital imaging applications.
Femtosecond lasers are very effective tools for three-dimensional micromachining of transparent materials. Nonlinear absorption of tightly focused femtosecond laser pulses allows energy to be deposited in a micrometer-sized volume in the bulk of the sample. If enough energy is deposited, localized changes in the material are produced (a change in refractive index, for example). These localized changes are the building blocks from which three-dimensional structures can be produced. With sufficiently tight focusing, the threshold for producing these changes can be achieved with pulse energies that are available directly from laser oscillators, offering greatly increased machining speeds and simpler, cheaper technology compared to using amplified lasers. In addition, the inter-pulse spacing from a laser oscillator is much shorter than the time required for energy deposited by one pulse to diffuse out of the focal volume. As a result, irradiation with multiple pluses on one spot in the sample leads to an accumulation of heat around the focal region. This localized heating provides another mechanism by which material properties can be altered. We demonstrate the three-dimensional fabrication of optical waveguides and microfluidic channels using pulse energies of only a few nanojoules to tens of nanojoules.
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