Evan P. Perillo

Bubble-Pen Lithography

Current lithography techniques, which employ photon, electron, or ion beams to induce chemical or physical reactions for micro/nano-fabrication, have remained challenging in patterning chemically synthesized colloidal particles, which are emerging as building blocks for functional devices. Herein, we develop a new technique - bubble-pen lithography (BPL) - to pattern colloidal particles on substrates using optically controlled microbubbles. Briefly, a single laser beam generates a microbubble at the interface of colloidal suspension and a plasmonic substrate via plasmon-enhanced photothermal effects. The microbubble captures and immobilizes the colloidal particles on the substrate through coordinated actions of Marangoni convection, surface tension, gas pressure, and substrate adhesion. Through directing the laser beam to move the microbubble, we create arbitrary single-particle patterns and particle assemblies with different resolutions and architectures. Furthermore, we have applied BPL to pattern CdSe/ZnS quantum dots on plasmonic substrates and polystyrene (PS) microparticles on two-dimensional (2D) atomic-layer materials. With the low-power operation, arbitrary patterning and applicability to general colloidal particles, BPL will find a wide range of applications in microelectronics, nanophotonics, and nanomedicine.

Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination

Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 μm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 μm. The system has a response time of 1 ms with a temporal resolution down to 50 μs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 μm in tumour spheroids.

Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser.

Here we demonstrate that a mode-locked ytterbium fiber laser for two-photon fluorescence microscopy can be built for

Molecular-Fluorescence Enhancement via Blue-Shifted Plasmon-Induced Resonance Energy Transfer

13,000. The laser emits at a wavelength of 1060 nm with a usable average power of 1 W at a repetition rate of 40 MHz and a compressed pulse width of 81 fs at the sample. The laser is used to obtain deep in vivo two-color images of layer-V pyramidal neurons expressing YFP and vasculature labelled with Texas Red at depths up to 900 µm. The sub-1 µm features of dendritic spines can be resolved at a 200 µm depth.

Regioselective Localization and Tracking of Biomolecules on Single Gold Nanoparticles

We report molecular-fluorescence enhancement via the blue-shifted plasmon-induced resonance energy transfer (PIRET) from single Au nanorods (AuNRs) to merocyanine (MC) dye molecules. The blue-shifted PIRET occurs when there is a proper spectral overlap between the scattering of AuNRs and the absorption of MC molecules. Along with the quenching of scattering from AuNRs, the blue-shifted PIRET enhances the fluorescence of nearby molecules. On the basis of the fluorescence enhancement, we conclude that AuNRs can be used as donors with clear advantages to excite the fluorescence of molecules as acceptors in AuNR-molecule hybrids. On the one hand, compared to conventional molecular donors in Förster resonance energy transfer (FRET), AuNRs have much larger absorption cross sections at the plasmon resonance frequencies. On the other hand, energy-transfer efficiency of PIRET decreases at a lower rate than that of FRET when the donor-acceptor distance is increased. Besides, the blue-shifted PIRET allows excitation with incident light of lower energy than the acceptors absorption, which is difficult to achieve in FRET because of the Stokes shift. With the capability of enhancing molecular fluorescence with excitation light of low intensity and long wavelength, the blue-shifted PIRET will expand the applications of nanoparticle- molecule hybrids in biosensing and bioimaging by increasing signal-to-noise ratio and by reducing photodamage to biological cells and organelles at the targeted areas.

3D single-molecule tracking using one- and two-photon excitation microscopy

Selective localization of biomolecules at the hot spots of a plasmonic nanoparticle is an attractive strategy to exploit the light–matter interaction due to the high field concentration. Current approaches for hot spot targeting are time‐consuming and involve prior knowledge of the hot spots. Multiphoton plasmonic lithography is employed to rapidly immobilize bovine serum albumin (BSA) hydrogel at the hot spot tips of a single gold nanotriangle (AuNT). Regioselectivity and quantity control by manipulating the polarization and intensity of the incident laser are also established. Single AuNTs are tracked using dark‐field scattering spectroscopy and scanning electron microscopy to characterize the regioselective process. Fluorescence lifetime measurements further confirm BSA immobilization on the AuNTs. Here, the AuNT‐BSA hydrogel complexes, in conjunction with single‐particle optical monitoring, can act as a framework for understanding light–molecule interactions at the subnanoparticle level and has potential applications in biophotonics, nanomedicine, and life sciences.

Improving z-tracking accuracy in the two-photon single-particle tracking microscope

Three dimensional single-molecule tracking (3D-SMT) has revolutionized the way we study fundamental cellular processes. By analyzing the spatial trajectories of individual molecules (e.g. a receptor or a signaling molecule) in 3D space, one can discern the internalization or transport dynamics of these molecules, study the heterogeneity of subcellular structures, and elucidate the complex spatiotemporal regulation mechanisms. Sub-diffraction localization precision, sub-millisecond temporal resolution and tens-of-seconds observation period are the benchmarks of current 3D-SMT techniques. We have recently built two molecular tracking systems in our labs. The first system is a previously reported confocal tracking system, which we denote as the 1P-1E-4D (one-photon excitation, one excitation beam, and four fiber-coupled detectors) system. The second system is a whole new design that is based on two-photon excitation, which we denote as the 2P-4E-1D (two-photon excitation, four excitation beams, and only one detector) system. Here we compare these two systems based on Monte Carlo simulation of tracking a diffusing fluorescent molecule. Through our simulation, we have characterized the limitation of individual systems and optimized the system parameters such as magnification, z-plane separation, and feedback gains.

High-Resolution Bubble Printing of Quantum Dots

Here, we present a method that can improve the z-tracking accuracy of the recently invented TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) microscope. This method utilizes a maximum likelihood estimator (MLE) to determine the particles 3D position that maximizes the likelihood of the observed time-correlated photon count distribution. Our Monte Carlo simulations show that the MLE-based tracking scheme can improve the z-tracking accuracy of TSUNAMI microscope by 1.7 fold. In addition, MLE is also found to reduce the temporal correlation of the z-tracking error. Taking advantage of the smaller and less temporally correlated z-tracking error, we have precisely recovered the hybridization-melting kinetics of a DNA model system from thousands of short single-particle trajectories in silico. Our method can be generally applied to other 3D single-particle tracking techniques.

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection

Semiconductor quantum dots (QDs) feature excellent properties, such as high quantum efficiency, tunable emission frequency, and good fluorescence stability. Incorporation of QDs into new devices relies upon high-resolution and high-throughput patterning techniques. Herein, we report a new printing technique known as bubble printing (BP), which exploits a light-generated microbubble at the interface of colloidal QD solution and a substrate to directly write QDs into arbitrary patterns. With the uniform plasmonic hot spot distribution for high bubble stability and the optimum light-scanning parameters, we have achieved full-color QD printing with submicron resolution (650 nm), high throughput (scanning rate of ∼10-2 m/s), and high adhesion of the QDs to the substrates. The printing parameters can be optimized to further control the fluorescence properties of the patterned QDs, such as emission wavelength and lifetime. The patterning of QDs on flexible substrates further demonstrates the wide applicability of this new technique. Thus, BP technique addresses the barrier of achieving a widely applicable, high-throughput and user-friendly patterning technique in the submicrometer regime, along with simultaneous fluorescence modification capability.

Single particle tracking through highly scattering media with multiplexed two-photon excitation

In the past two decades, significant advances have been made in single-molecule detection which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics, and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.