Laser-free Ultrafast Tunable Stroboscopic TEM imaging for Biomedical Applications

Title: Laser-free Ultrafast Tunable Stroboscopic TEM imaging for Biomedical Applications PI: C. Jing Project Summary/Abstract Major advances in cell biology and biomedical research are tightly linked to innovations in microscopy. Modern cryogenic transmission electron microscopy (cryo-EM) achieves near-atomic-resolution images but faces key barriers. Beam-induced radiation damage during image exposure limits higher resolution: useful signal added per incident electron decreases due to damage, while added noise remains roughly constant, meaning an optimum exposure exists beyond which signal-to-noise worsens. Cryotomographic techniques allow 3D imaging, but their tilted images can suffer from vibrational blur. And ultrafast transmission electron microscopy (UTEM) now takes “molecular movies” rather than static images by using femtosecond lasers, but with the exorbitant expense and invasive modifications; only a handful exist worldwide and image acquisition is slow due to typical 1MHz or lower laser repetition rates. Euclid Beamlabs, LLC, is addressing all these challenges by developing a single technology: a laser-free, cost-effective, retrofittable TEM pulser, widely tunable from Herz to Gigahertz repetition rate and microsecond to picosecond pulse duration. Our first-generation pulser won a 2019 Randamp;D 100 Award. The NIH SBIR Phase I project from July to November 2019 demonstrated first bio-imaging on two Euclid-retrofitted JEOL TEMs. We showed irradiation damage mitigation of C36H74 paraffin and purple membrane: compared to continuous beam, our GHz pulsed beam produced up to 2.5x less irradiation damage at equal dose, repeatably tested to 10 electrons per square Angstrom (10e-/Å2) at both 200 and 300 kV. Phase II will build on the successes of Phase I. We will introduce second-generation pulser technology in a cryo-TEM for the first time. The dynamic range of pulse duty factor will be ten orders of magnitude higher than in Phase I using our newly patented Pulse Picker, an essential requirement to address the previously introduced cryo-EM imaging limitations. We will produce a pulsed beam suitable for vibration-insensitive cryotomography, potentially allowing sharper images and full tilt-series in seconds. We will optimize pulse structure for reduced radiation damage and higher critical dose, leading to higher contrast and higher resolution. We will assess whether a pulsed beam also improves vitreous water crystallization, a canonical cryo-EM limitation. And we will quantify temperature- and pulse-dependent radiation damage interrelationships for crystalline and single-particle bio-samples. At the conclusion of Phase II, the new pulsed cryo-EM and the many proof-of-principle use cases will initiate commercialization of this affordable, retrofittable, versatile system to bring high resolution, high contrast, vibration-insensitive, and time-resolved electron microscopy to the wider bio- imaging community.Project Narrative Euclid Beamlabs, LLC is developing a powerful new beam-pulser technology for biomedical and cell imaging on conventional and cryogenic transmission electron microscopes (TEM and cryo- EM) to enable picosecond “molecular video electron microscopy” and reveal how biomacromolecules move and function. Unlike past laser-based approaches, our laser-free concept is nearly a million dollars less expensive; it is retrofittable into legacy TEMs already owned by universities, hospitals, and research centers; and it can illuminate thousands of images in the same time a laser-based system takes just one. Moreover, our “molecular video” timing is tunable by up to a trillion times, opening exciting new paradigms for images with less blur, higher contrast, and incredibly sharp resolution to help advance the future of biology, bio-materials, and biomedicine.