SBIR-STTR Award

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.

Public Health Relevance Statement:
Project narrative To study the brain, we need to be able to image the 3D interaction of neurons at a fast millisecond timescale. However, current 3D microscope scans rely on mechanical methods that are about 15x too slow. We will introduce a nonmechanical millisecond-timescale 3D scan method that uses liquid crystal polarization grating (LCPG) lenses.

Project Terms:
Back; base; Brain; Caliber; Characteristics; Custom; Development; Devices; Electrodes; Evaluation; Feedback; Financial compensation; Image; imaging system; indexing; Length; lens; Licensing; Light; Link; liquid crystal; Liquid substance; Location; Measurement; Mechanics; Methods; Microscope; Microscopy; millisecond; Motion; Movement; neuronal circuitry; Neurons; Performance; performance tests; Phase; Positioning Attribute; Procedures; Process; response; Sampling; Scanning; Speed; Spottings; Staging; System; Techniques; Technology; Testing; Thick; transmission process; two-photon; vibration

This Phase II Lab-to-Marketplace proposal aims to commercialize a new remote focusing technique that can change the focus of a microscope by as much as 500 ?m in less than 40 ?s, 3 orders of magnitude faster than other discrete focus change techniques. Our initial market is neuroscience imaging, where the ability of researchers to step between focal planes at the millisecond timescale of neuronal circuits is limited by the speed and/or complexity of current remote focusing techniques. Piezo translated objectives and liquid electrically tunable lenses have fairly long settling times, on the order of 10-20 ms, which lowers the effective duty cycle at high frame rate imaging. When these devices are operated in resonant mode, duty cycles are higher, but there are still long delays between accessing disparate axial regions. Our remote focusing device uses thin liquid crystal (LC) switches and liquid crystal polarization gratings (LCPGs) to create dynamic lenses. We originally introduced LCPGs as linear gratings for nonmechanical multiangle beamsteering, but realized they can also be leveraged for extremely high speed focusing. In Phase I, we demonstrated the first use of LC switches and circularly-patterned LCPG lenses for changing the focus of a multiphoton microscope system. We were able to shift the focus by ~300 micrometers in < 40 ?s; the settling time is independent of the device’s diameter or of the distance shifted. Axial focusing at these deeply submillisecond timescales is crucial in particular for imaging 3D neural circuits, but will also find applications in other areas where speed and/or hysteresis-free reproducibility is important. In Phase II we plan to bring the LCPG remote focusing lens stack to market with a target price of $1000 and an initial target application of optogenetics research. To reach this target price, we will undertake a systematic process development effort to increase yield, similar to techniques we have used in the LC microdisplay industry. We will also develop an in-house custom LC switch controller for greatly reduced cost and increased robustness and ease of use. With a new grating recording setup we will be able to record LCPGs with 50 mm diameter, and also address wavefront error. With our collaborators at Columbia University, we will characterize the PSF, magnification, dispersion, and wavelength-dependent signal-to-noise ratio in multiple commercial and homebuilt multiphoton microscopes, and with multiple microscope objectives. We will perform interlaminar, intralaminar, and multiplane imaging in live, behaving mice as demonstrations of the new capabilities enabled by this fast remote focusing device.

Public Health Relevance Statement:
PROJECT NARRATIVE We will commercialize a fast remote focusing lens to allow brain researchers to study 3D neural interconnections. This lens, based on a nonmechanical beamsteering method called liquid crystal polarization grating stacks, can change focus at timescales 3 orders of magnitude faster than other discrete focusing methods. This will enable new progress in brain imaging and optogenetics.

Project Terms:
Address; Area; awake; base; Brain; Brain imaging; Caliber; cost; Custom; Dependence; Dependency; design; Development; Devices; experimental study; flexibility; Functional Imaging; Image; imaging system; Industry; Length; lens; Light; liquid crystal; Liquid substance; Methods; Microscope; millisecond; Modeling; Modernization; Mus; neural circuit; Neural Interconnection; neuronal circuitry; Neurons; Neurosciences; next generation; Noise; optogenetics; Pattern; Performance; Phase; photoactivation; Price; Process; Publications; Pupil; Reproducibility; Research; Research Personnel; response; Running; Sampling; Signal Transduction; Speed; System; Techniques; Testing; Thinness; Time; Translating; two-photon; Universities; Voice