Bowman Lab

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult . Lifetime resolutions of <5 picoseconds at 1 kilohertz were achieved for single-cell voltage recordings. Lifetime readout is limited by photon shot noise, and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Nanosecond electron pulses are appealing for ultrafast imaging and electron-gating applications, where tunable currents and narrow energy spreads are desirable. Here, we demonstrate photoemission from a Schottky emitter triggered by nanosecond laser pulses. Using photon energies optimally tuned to the emission potential barrier, we generate pulses containing over 105 electrons with energy spreads below 1 eV by means of a prompt single-photon photoemission process. These results are consistent with a theoretical model of laser-triggered electron emission and energetic broadening during propagation and can be widely implemented.

We demonstrate an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently gate images at 39 MHz, allowing fluorescence lifetime to be captured on standard camera sensors. Lifetime imaging of single molecules is enabled in wide field with exposure times of less than 100 ms. This capability allows combination of wide-field FLIM with single-molecule super-resolution localization microscopy. Fast single-molecule dynamics such as FRET and molecular binding events are captured from wide-field images without prior spatial knowledge. A lifetime sensitivity of 1.9 times the photon shot-noise limit is achieved, and high throughput is shown by acquiring wide-field FLIM images with millisecond exposure and >10 photons per frame. Resonant electro-optic FLIM allows lifetime contrast in any wide-field microscopy method.

We present the design and prototype of a switchable electron mirror, along with a technique for driving it with an arbitrary pulse shape. We employ a general technique for electronic pulse-shaping, where high fidelity of the pulse shape is required, but the characteristics of the system, which are possibly nonlinear, are not known. This driving technique uses an arbitrary waveform generator to pre-compensate the pulse, with a simple iterative algorithm used to generate the input waveform. This is a broadly applicable, general method for arbitrary pulse shaping. Driving our switchable electron mirror with a flat-top pulse, we demonstrate an improvement in rms error of roughly two orders of magnitude compared to an uncompensated waveform. Our results demonstrate the feasibility of high fidelity waveform reproduction in the presence of nonidealities, with immediate applications in the realization of novel electron optical components.

We study electron emission from a Schottky tip induced by nanosecond laser pulses. Prompt sub-nanosecond emission is observed at low laser power, with moderate voltage bias applied to the tip. We show that electron pulses demonstrate high extinction with electron emission after the pulse suppressed by up to 92 dB. Photoemission is shown to be highly nonlinear with laser intensity while maintaining nearly linear field emission, as probed by the laser polarization dependence. We suggest the emission is described by a photo-assisted thermally enhanced field emission process.

Multi-pass transmission electron microscopy (MPTEM) has been proposed as a way to reduce damage to radiation-sensitive materials. For the field of cryo-electron microscopy (cryo-EM), this would significantly reduce the number of projections needed to create a 3D model and would allow the imaging of lower-contrast, more heterogeneous samples. We have designed a 10 keV proof-of-concept MPTEM. The column features fast-switching gated electron mirrors which cause each electron to interrogate the sample multiple times. A linear approximation for the multi-pass contrast transfer function (CTF) is developed to explain how the resolution depends on the number of passes through the sample.

Nanosecond temporal resolution enables new methods for wide-field imaging like time-of-flight, gated detection, and fluorescence lifetime. The optical efficiency of existing approaches, however, presents challenges for low-light applications common to fluorescence microscopy and single-molecule imaging. We demonstrate the use of Pockels cells for wide-field image gating with nanosecond temporal resolution and high photon collection efficiency. Two temporal frames are obtained by combining a Pockels cell with a pair of polarizing beam-splitters. We show multi-label fluorescence lifetime imaging microscopy (FLIM), single-molecule lifetime spectroscopy, and fast single-frame FLIM at the camera frame rate with 10-10 times higher throughput than single photon counting. Finally, we demonstrate a space-to-time image multiplexer using a re-imaging optical cavity with a tilted mirror to extend the Pockels cell technique to multiple temporal frames. These methods enable nanosecond imaging with standard optical systems and sensors, opening a new temporal dimension for wide-field low-light microscopy.

We present a simple apparatus for improved surface modification of polydimethylsiloxane (PDMS) microfluidic devices. A single treatment chamber for plasma activation and chemical/physical vapor deposition steps minimizes the time-dependent degradation of surface activation that is inherent in multi-chamber techniques. Contamination and deposition irregularities are also minimized by conducting plasma activation and treatment phases in the same vacuum environment. An inductively coupled plasma driver allows for interchangeable treatment chambers. Atomic force microscopy confirms that silane deposition on PDMS gives much better surface quality than standard deposition methods, which yield a higher local roughness and pronounced irregularities in the surface.