Research Project
9255237
Project summary The fast millisecond timescale of neuronal activity has posed a difficulty for 3D volumetric imaging, whose speed is limited in part by the axial scan methods currently available. The use of electrically tunable lenses (ETLs) for remote focusing confers speed and vibration-reduction advantages over the more traditional sample stage or microscope objective motion, but current state-of-the-art ETLs are liquid lenses that still require mechanical movement from a piezo ring, with their transition speed limited to ~15ms by mechanical ringing. We propose to build and test an ETL based on switchable liquid crystal polarization grating lenses (LCPG lenses) that can perform remote axial focusing at 1ms timescale, an order of magnitude speed improvement over state-of-the-art axial focusing techniques. The LCPG lens is a nonmechanical device and thus has no ringing or hysteresis, particularly useful for repeated scans of the same sample location, or for superresolution techniques where absolute repeatability in axial position is key. The speed of the LCPG lens is not linked to its aperture size, and LCPG lenses can be easily made with 25mm or larger apertures, avoiding vignetting by matching or exceeding the back aperture diameters of modern high-performance objectives. Unlike a piezo- liquid ETL, a LCPG lens can be made with any custom lens profile, including aspherical, and can include compensation for aberrations. Although the LCPG lens offers discrete rather than continuous focal scanning, these devices have >99% efficiency and can be stacked to produce as many focal planes as desired. Using 0.2mm substrates, an 8-stage LCPG lens would be just 4.8mm thick, but could achieve 256 focal planes. The Phase I LCPG lens will have 2 stages and 3 available focal planes, with clear aperture and focal plane location tailored for use as a high-speed remote focusing lens in a two-photon (2P) microscope system. Specific aims: 1. LCPG lens fabrication: Includes fabrication of both LCPG lenses and liquid crystal waveplate switches, assembly into a cascaded stack, index matching, and addition of electrodes. 2. LCPG lens characterization: Includes benchtop characterization of efficiency at the target wavelength, switching speed, and Shack-Hartmann measurement of wavefront quality compared to the template lens. 3. Integration into CW microscope: Using one of our existing CW microscope + 2D spatial light modulator systems, we will characterize the amount of focal length shift introduced by the LCPG lens, along with the 3D focal spot sizes and aberrations in different focal planes. 4. Integration into 2P microscope: We will directly compare the performance of the LCPG lens to the state- of-the-art piezo-liquid lens by replacing a piezo-liquid ETL with the Phase I lens in a 2P microscope system used for 3D neuronal imaging. We will gather feedback from this end user system to focus our further development efforts.
