Limor Minai

High levels of reactive oxygen species in gold nanoparticle-targeted cancer cells following femtosecond pulse irradiation

Cancer cells could be locally damaged using specifically targeted gold nanoparticles and laser pulse irradiation, while maintaining minimum damage to nearby, particle-free tissue. Here, we show that in addition to the immediate photothermal cell damage, high concentrations of reactive oxygen species (ROS) are formed within the irradiated cells. Burkitt lymphoma B cells and epithelial breast cancer cells were targeted by antibody-coated gold nanospheres and irradiated by a few resonant femtosecond pulses, resulting in significant elevation of intracellular ROS which was characterized and quantified using time-lapse microscopy of different fluorescent markers. The results suggest that techniques that involve targeting of various malignancies using gold nanoparticles and ultrashort pulses may be more effective and versatile than previously anticipated, allowing diverse, highly specific set of tools for local cancer therapy.

Noninvasive imaging of flowing blood cells using label-free spectrally encoded flow cytometry

Optical microscopy of blood cells in vivo provides a unique opportunity for clinicians and researchers to visualize the morphology and dynamics of circulating cells, but is usually limited by the imaging speed and by the need for exogenous labeling of the cells. Here we present a label-free approach for in vivo flow cytometry of blood using a compact imaging probe that could be adapted for bedside real-time imaging of patients in clinical settings, and demonstrate subcellular resolution imaging of red and white blood cells flowing in the oral mucosa of a human volunteer. By analyzing the large data sets obtained by the system, valuable blood parameters could be extracted and used for direct, reliable assessment of patient physiology.

Controlled release of Rituximab from gold nanoparticles for phototherapy of malignant cells.

Releasing drug molecules at their targets with high spatial and temporal accuracy could aid numerous clinical applications which require low systemic damage and low side effects. Nano-carriers of drugs are an attractive solution for such task, allowing specific accumulation in tumors and gradual release of their payload. Here, we utilize gold nanospheres conjugated to Rituximab, an anti-CD20 monoclonal antibody-based drug, for carrying and releasing the drug upon irradiation of specifically tailored femtosecond laser pulses. The released anti-CD20 molecules retain their functionality and ability of triggering the complement-dependent cytotoxicity. This effect comes in addition to cell necrosis caused by the plasmonic nanometric shock waves emanating from the nanospheres and rupturing the plasma membranes. Main advantages of the presented technique include high spatial and temporal resolution, low toxicity and high repeatability and consistency due to the morphological stability of the nanospheres.

Optical Nanomanipulations of Malignant Cells: Controlled Cell Damage and Fusion

Specifically targeting and manipulating living cells is a key challenge in biomedicine and in cancer research in particular. Several studies have shown that nanoparticles irradiated by intense lasers are capable of conveying damage to nearby cells for various therapeutic and biological applications. In this work ultrashort laser pulses and gold nanospheres are used for the generation of localized, nanometric disruptions on the membranes of specifically targeted cells. The high structural stability of the nanospheres and the resonance pulse irradiation allow effective means for controlling the induced nanometric effects. The technique is demonstrated by inducing desired death mechanisms in epidermoid carcinoma and Burkitt lymphoma cells, and initiating efficient cell fusion between various cell types. Main advantages of the presented approach include low toxicity, high specificity, and high flexibility in the regulation of cell damage and cell fusion, which would allow it to play an important role in various future clinical and scientific applications.

Multiple-channel spectrally encoded imaging

Spectrally encoded endoscopy (SEE) uses miniature diffractive optics to encode space with wavelength, allowing video-rate three-dimensional imaging through sub-millimeter, flexible endoscopic probes. Here we present a new approach for SEE in which the illumination and the collection channels are separated in space, and spectral encoding is present only in the collection channel. Bench-top experiments using spatially incoherent white light illumination reveal significant improvement in image quality and considerable reduction of speckle noise compared to conventional techniques, and show that the new system is capable of high sensitivity fluorescence imaging of single cells. The presented new approach would allow improved functionality and usability of SEE.

Optically induced cell fusion using bispecific nanoparticles.

Redirecting the immune system to eliminate tumor cells is a promising alternative to traditional cancer therapies, most often requiring direct interaction between an immune system effector cell and its target. Herein, a novel approach for selective attachment of malignant cells to antigen-presenting cells by using bispecific nanoparticles is presented. The engaged cell pairs are then irradiated by a sequence of resonant femtosecond pulses, which results in widespread cell fusion and the consequent formation of hybrid cells. The dual role of gold nanoparticles as conjugating agents and fusion promoters offers a simple yet effective means for specific fusion between different cells. This technology could be useful for a variety of in vitro and in vivo applications that call for selective fusion between cells within a large heterogenic cell population.

Spectrally encoded spectral imaging

Spectral imaging, i.e. the acquisition of the spectrum emitted from each sample location, is a powerful tool for a wide variety of applications in science and technology. For biomedical applications, spectral imaging is important for accurate analysis of a biological specimen and for assisting clinical diagnosis, however it could be challenging mainly due to the typically low damage thresholds and strict time constraints. Here, we present a fiber-based technique termed spectrally encoded spectral imaging (SESI), in which a fully emitted spectrum is captured from each resolvable point of a specimen using an additional lateral scanning of the spectrally encoded line. The technique is demonstrated by capturing spectral data cubes of a color print and of a green leaf, and its potential advantage in signal-to-noise ratio is theoretically discussed. Using a miniaturized grating-lens configuration, SESI could be conducted endoscopically, allowing minimally invasive color and spectral imaging in remote locations of the body.

Experimental Proof for the Role of Nonlinear Photoionization in Plasmonic Phototherapy

Targeting individual cells within a heterogeneous tissue is a key challenge in cancer therapy, encouraging new approaches for cancer treatment that complement the shortcomings of conventional therapies. The highly localized interactions triggered by focused laser beams promise great potential for targeting single cells or small cell clusters; however, most laser-tissue interactions often involve macroscopic processes that may harm healthy nearby tissue and reduce specificity. Specific targeting of living cells using femtosecond pulses and nanoparticles has been demonstrated promising for various potential therapeutic applications including drug delivery via optoporation, drug release, and selective cell death. Here, using an intense resonant femtosecond pulse and cell-specific gold nanorods, we show that at certain irradiation parameters cell death is triggered by nonlinear plasmonic photoionization and not by thermally driven processes. The experimental results are supported by a physical model for the pulse-particle-medium interactions. A good correlation is found between the calculated total number and energy of the generated free electrons and the observed cell death, suggesting that femtosecond photoionization plays the dominant role in cell death.

Plasmonic targeting of cancer cells in a three-dimensional natural hydrogel

Using specifically designed gold nanoparticles and local laser irradiation, individual cells and small cell clusters could be targeted on a microscopic scale with minimal toxicity to nearby tissue. To date, most scientific studies and technological demonstrations of this approach were conducted on two-dimensional cultures, while most feasibility tests and preclinical trials were conducted using animal models. For bridging the gap between two-dimensional cell cultures and animal experiments, we propose and demonstrate the use of a natural hydrogel for studying the effect of intense, ultrashort laser pulses on a gold nanoparticle targeted tissue. Using illumination parameters comparable to those used with two-dimensional cultures, we show the complete eradication of multilayered cell colonies comprising normal fibroblasts and malignant epithelial cells co-cultured on a hydrogel scaffold. By evaluating the extent of cell damage for various pulse durations at off-resonance irradiation, we find that the observed damage mechanism was dominated by rapid thermal transitions around the gold nanospheres, rather than by photoionization. The work provides a new tool for understanding the complex pulse-particle-tissue interactions and demonstrates the important role of nanoparticle mediated cavitation bubbles in a thick, multilayered tissue.

Plasmonic eradication of malignant cells in a three dimensional culture (Conference Presentation)

The interaction between gold nanoparticles and femtosecond laser irradiation has been shown to be effective in damaging malignant cells in two-dimensional cell cultures. Further studies with rodent models have also shown promising results; however, rodent physiology and genetics are known to differ substantially from that of humans, often failing to successfully predict the human response to treatment. For bridging the gap between two-dimensional cell cultures and animal models for plasmonic phototherapy, we use a natural hydrogel extracted from mouse sarcoma as a three dimensional scaffold for growing breast cancer epithelial cells in co-culture with healthy fibroblast cells. The malignant cells were specifically targeted using anti-EGFR-coated gold nanospheres, followed by washout of unbound particles and irradiation by amplified femtosecond pulses at off-resonance wavelength of 800 nm. Irradiated cell colonies seized to develop and grow; after approximately three days, time-lapse imaging revealed widespread death of both normal fibroblasts and malignant epithelial cells, leading to disintegration of all cell colonies. Pulse broadening experiments demonstrated similar cell death for pulses between 45 fs and 400 fs of equal energy, while pulses longer than 500 fs had no visible effect on the cells. The results suggest that cell damage is primarily photothermal, and that cells in three-dimensional cultures are more resistant to the effect of the laser pulses, requiring several irradiation sequences and exhibiting slow colony disintegration.