Biqin Dong

A transparent broadband ultrasonic detector based on an optical micro-ring resonator for photoacoustic microscopy

Photoacoustic microscopy (PAM) does not rely on contrast agent to image the optical absorption contrast in biological tissue. It is uniquely suited for measuring several tissue physiological parameters, such as hemoglobin oxygen saturation, that would otherwise remain challenging. Researchers are designing new clinical diagnostic tools and multimodal microscopic systems around PAM to fully unleash its potential. However, the sizeable and opaque piezoelectric ultrasonic detectors commonly used in PAM impose a serious constraint. Our solution is a coverslip-style optically transparent ultrasound detector based on a polymeric optical micro-ring resonator (MRR) with a total thickness of 250 μm. It enables highly-sensitive ultrasound detection over a wide receiving angle with a bandwidth of 140 MHz, which corresponds to a photoacoustic saturation limit of 287 cm−1, at an estimated noise-equivalent pressure (NEP) of 6.8 Pa. We also established a theoretical framework for designing and optimizing the MRR for PAM.

Photoacoustic probe using a microring resonator ultrasonic sensor for endoscopic applications

We designed an all-optical photoacoustic (PA) probe for endoscopic applications by employing an optically transparent, coverslip-type, polymeric microring resonator ultrasonic sensor. We experimentally quantified the axial, tangential, and radial resolutions and angular sensitive stability of this probe. Using this probe, we achieved volumetric imaging of several phantoms. Our all-optical probe design offers clear benefit in integrating PA endoscope with other optical endoscopic imaging modalities to facilitate the transformation from bench to bedside.

Isometric multimodal photoacoustic microscopy based on optically transparent micro-ring ultrasonic detection

Photoacoustic microscopy (PAM) is an attractive imaging tool complementary to established optical microscopic modalities by providing additional molecular specificities through imaging optical absorption contrast. While the development of optical resolution photoacoustic microscopy (ORPAM) offers high lateral resolution, the acoustically-determined axial resolution is limited due to the constraint in ultrasonic detection bandwidth. ORPAM with isometric spatial resolution along both axial and lateral direction is yet to be developed. Although recently developed sophisticated optical illumination and reconstruction methods offer improved axial resolution in ORPAM, the image acquisition procedures are rather complicated, limiting their capabilities for high-speed imaging and being easily integrated with established optical microscopic modalities. Here we report an isometric ORPAM based on an optically transparent micro-ring resonator ultrasonic detector and a commercial inverted microscope platform. Owing to the superior spatial resolution and the ease of integrating our ORPAM with established microscopic modalities, single cell imaging with extrinsic fluorescence staining, intrinsic autofluorescence, and optical absorption can be achieved simultaneously. This technique holds promise to greatly improve the accessibility of PAM to the broader biomedical researchers.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Significance Glioblastoma is a deadly brain tumor with no cure. Brain tumor-initiating cells (BTICs) have been recognized as the key driver behind the unstoppable malignant growth, therapy resistance, and recurrence. BTICs are exceptionally difficult to target because of heterogeneous genetic and epigenetic aberrations that are challenging to reverse therapeutically using conventional pharmaceuticals or biologics. Here we report a lipopolymeric nanoparticle (LPNP) formulation that demonstrates a surprisingly high affinity for BTICs and the capacity to encapsulate multiple siRNAs for potent and targeted anti-BTIC therapy. We show that direct infusion of LPNP siRNAs to brain tumors effectively impedes tumor growth in mouse and provides encouraging survival benefits. This multiplexed nanomedicine platform carries strong potential for personalized anti-BTIC therapies. Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood–brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Optical Detection of Ultrasound in Photoacoustic Imaging

Objective: Photoacoustic (PA) imaging emerges as a unique tool to study biological samples based on optical absorption contrast. In PA imaging, piezoelectric transducers are commonly used to detect laser-induced ultrasonic waves. However, they typically lack adequate broadband sensitivity at ultrasonic frequency higher than 100 MHz, whereas their bulky size and optically opaque nature cause technical difficulties in integrating PA imaging with conventional optical imaging modalities. To overcome these limitations, optical methods of ultrasound detection were developed and shown their unique applications in PA imaging. Methods: We provide an overview of recent technological advances in optical methods of ultrasound detection and their applications in PA imaging. A general theoretical framework describing sensitivity, bandwidth, and angular responses of optical ultrasound detection is also introduced. Results: Optical methods of ultrasound detection can provide improved detection angle and sensitivity over significantly extended bandwidth. In addition, its versatile variants also offer additional advantages, such as device miniaturization, optical transparency, mechanical flexibility, minimal electrical/mechanical crosstalk, and potential noncontact PA imaging. Conclusion: The optical ultrasound detection methods discussed in this review and their future evolution may play an important role in PA imaging for biomedical study and clinical diagnosis.

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

Significance Fluorescence photoswitching of native, unmodified deoxyribonucleic acid (DNA) using visible light facilitates the label-free nanoscale imaging of chromatin structures based on the principle of single-molecule photon localization microscopy (PLM). With a demonstrated sub–20-nm resolution, DNA-PLM provides an ideal technique to visualize the spatial organization of single or groups of nucleosomes and quantitatively estimate the nucleosome occupancy level of DNA in unstained chromosomes and nuclei. This study paves a way for revealing nanoscopic features of chromatin without the need for exogenous labels and could substantially expand our understanding of the structure–function relationship of chromatin. Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.

Super-resolution spectroscopic microscopy via photon localization

Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm—a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

Isolating single cells in a neurosphere assay using inertial microfluidics

Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Deans force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (>90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays.

Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures

Optical imaging has offered unique advantages in material researches, such as spectroscopy and lifetime measurements of deeply embedded materials, which cannot be matched using electron or scanning-probe microscopy. Unfortunately, conventional optical imaging cannot provide the spatial resolutions necessary for many nanoscopic studies. Despite recent rapid progress, super-resolution optical imaging has yet to be widely applied to non-biological materials. Herein we describe a method for nanoscopic optical imaging of buried polymer nanostructures without the need for extrinsic staining. We observed intrinsic stochastic fluorescence emission or blinking from unstained polymers and performed spatial-temporal spectral analysis to investigate its origin. We further applied photon localization super-resolution imaging reconstruction to the detected stochastic blinking, and achieved a spatial resolution of at least 100 nm, which corresponds to a six-fold increase over the optical diffraction limit. This work demonstrates the potential for studying the static heterogeneities of intrinsic polymer molecular-specific properties at sub-diffraction-limited optical resolutions.

Quantitative Imaging of Rapidly Decaying Evanescent Fields Using Plasmonic Near-Field Scanning Optical Microscopy

Non-propagating evanescent fields play an important role in the development of nano-photonic devices. While detecting the evanescent fields in far-field can be accomplished by coupling it to the propagating waves, in practice they are measured in the presence of unwanted propagating background components. It leads to a poor signal-to-noise ratio and thus to errors in quantitative analysis of the local evanescent fields. Here we report on a plasmonic near-field scanning optical microscopy (p-NSOM) technique that incorporates a nanofocusing probe for adiabatic focusing of propagating surface plasmon polaritons at the probe apex, and for enhanced coupling of evanescent waves to the far-field. In addition, a harmonic demodulation technique is employed to suppress the contribution of the background. Our experimental results show strong evidence of background free near-field imaging using the new p-NSOM technique. Furthermore, we present measurements of surface plasmon cavity modes, and quantify their contributing sources using an analytical model.