Emilia Moradi

Modular Construction of Multifunctional Bioresponsive Cell-Targeted Nanoparticles for Gene Delivery

Multifunctional and modular block copolymers prepared from biocompatible monomers and linked by a bioreducible disulfide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block copolymer architecture enabled well-defined nanoparticles to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folate-receptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to nonbioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tunable way.

Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells

At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

High resolution 3D imaging of living cells with sub-optical wavelength phonons

Label-free imaging of living cells below the optical diffraction limit poses great challenges for optical microscopy. Biologically relevant structural information remains below the Rayleigh limit and beyond the reach of conventional microscopes. Super-resolution techniques are typically based on the non-linear and stochastic response of fluorescent labels which can be toxic and interfere with cell function. In this paper we present, for the first time, imaging of live cells using sub-optical wavelength phonons. The axial imaging resolution of our system is determined by the acoustic wavelength (λa = λprobe/2n) and not on the NA of the optics allowing sub-optical wavelength acoustic sectioning of samples using the time of flight. The transverse resolution is currently limited to the optical spot size. The contrast mechanism is significantly determined by the mechanical properties of the cells and requires no additional contrast agent, stain or label to image the cell structure. The ability to breach the optical diffraction limit to image living cells acoustically promises to bring a new suite of imaging technologies to bear in answering exigent questions in cell biology and biomedicine.

Sub-100 nm resolution microscopy based on proximity projection grating scheme

Structured illumination microscopy (SIM) has been widely used in life science imaging applications. The maximum resolution improvement of SIM, compared to conventional bright field system is a factor of 2. Here we present an approach to structured illumination microscopy using the proximity projection grating scheme (PPGS), which has the ability to further enhance the SIM resolution without invoking any nonlinearity response from the sample. With the PPGS-based SIM, sub-100 nm resolution has been obtained experimentally, and results corresponding to 2.4 times resolution improvement are presented. Furthermore, it will be shown that an improvement of greater than 3 times can be achieved.

Thin-film transducers for the detection and imaging of Brillouin oscillations in transmission on cultured cells

Mechanical imaging and characterisation of biological cells has been a subject of interest for the last twenty years. Ultrasonic imaging based on the scanning acoustic microscope (SAM) and mechanical probing have been extensively reported. Large acoustic attenuation at high frequencies and the use of conventional piezo-electric transducers limit the operational frequency of a SAM. This limitation results in lower resolution compared to an optical microscope. Direct mechanical probing in the form of applied stress by contacting probes causes stress to cells and exhibits poor depth resolution. More recently, laser ultrasound has been reported to detect ultrasound in the GHz range via Brillouin oscillations on biological cells. This technique offers a promising new high resolution acoustic cell imaging technique. In this work, we propose, design and apply a thin-film based opto-acoustic transducer for the detection in transmission of Brillouin oscillations on cells. The transducer is used to generate acoustic waves, protect the cells from laser radiation and enhance signal-to-noise ratio (SNR). Experimental traces are presented in water films as well as images of the Brillouin frequency of phantom and fixed 3T3 fibroblast cells.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system’s performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.