Cell selective introduction of therapeutic agents remains a challenging problem. Cavitation-based therapies including ultrasound-induced sonoporation and laser-induced optoporation have led the way for novel approaches to provide the potential of sterility and cell selectivity compared with viral or biochemical counterparts. Acoustic streaming, shockwaves and liquid microjets associated with the cavitation dynamics are implicated in gene and drug delivery. These approaches, however, often lead to non-uniform and sporadic molecular uptake that lacks refined spatial control and suffers from a significant loss of cell viability. Here we demonstrate spatially controlled cavitation instigated by laser-induced breakdown of an optically trapped single gold nanoparticle. Our unique approach employs optical tweezers to trap a single nanoparticle, which when irradiated by a nanosecond laser pulse is subject to laser-induced breakdown followed by cavitation. Using this method for laser-induced cavitation, we can gain additional degrees of freedom for the cavitation process - the particle material, its size, and its position relative to cells or tissues. We show the energy breakdown threshold of gold nanoparticles of l00nm with a single nanosecond laser pulse at 532 nm is three orders of magnitude lower than that for water, which leads to gentle nanocavitation enabling single cell transfection. We optimize the shear stress to the cells from the expanding bubble to be in the range of 1-10 kPa for transfection by precisely positioning a trapped gold nanoparticle, and thus nanobubble, relative to a cell of interest. The method shows transfection of plasmid-DNA into individual mammalian cells with an efficiency of 75%.
Shear stress has been recognized as one of the biophysical methods by which to permeabilize plasma membranes of cells. In particular, high pressure transient hydrodynamic flows created by laser-induced cavitation have been shown to lead to the uptake of fluorophores and plasmid DNA. While the mechanism and dynamics of cavitation have been extensively studied using a variety of time-resolved imaging techniques, the cellular response to the cavitation bubble and cavitation induced transient hydrodynamic flows has never been shown in detail. We use time-resolved quantitative phase microscopy to study cellular response to laser-induced cavitation bubbles. Laser-induced breakdown of an optically trapped polystyrene nanoparticle (500nm in diameter) irradiated with a single nanosecond laser pulse at 532nm creates transient shear stress to surrounding cells without causing cell lysis. A bi-directional transient displacement of cytoplasm is observed during expansion and collapse of the cavitation bubble. In some cases, cell deformation is only observable at the microsecond time scale without any permanent change in cell shape or optical thickness. On a time scale of seconds, the cellular response to shear stress and cytoplasm deformation typically leads to retraction of the cellular edge most exposed to the flow, rounding of the cell body and, in some cases, loss of cellular dry mass. These results give a new insight into the cellular response to laser-induced shear stress and related plasma membrane permeabilization. This study also demonstrates that laser-induced breakdown of an optically trapped nanoparticle offers localized cavitation (70 μm in diameter), which interacts with a single cell.
Transgenic animals are an essential means for investigating genetic processes in vivo, and depend on efficient
delivery techniques to introduce exogenous genetic material into the organism, often at the zygote stage. In this
study, we demonstrate an optical approach to microinjection based on a holographic system using a spatial light
modulator and a Ti: Sapphire laser. This integrated system is capable of both optical orientation and injection of 60-
μm diameter Pomatoceros lamarckii (P.lamarckii) embryos. Individual blastomeres of P. lamarckii embryos were
optoinjected with varying sizes of dextran molecules and Propidium iodide using an 800-nm femtosecond laser with
controlled dosage. We also show that the technique is able to deliver materials to cells located deep within a welldeveloped
embryo. As a visual confirmation of successful optoinjection, the presence of gas bubbles was observed
as a function of laser power and exposure time. Small gas bubbles, less than 5-μm in diameter, were found to be
tolerated by the irradiated embryo. Furthermore, when switched to the continuous wave mode, the laser could exert
optical forces upon the embryo. This facilitated computer-controlled handling and orientation of P. lamarckii
embryos without compromising viability. Our multimodal optical platform offers a sterile, non-contact and robust
alternative to traditional microinjection. This work is a step towards applications in developmental biology such as
cell lineage mapping and formation of transgenic animals using an optical approach.
Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying
and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into
primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present
more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be
difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of
an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and
loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and
viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug
delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells;
however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an
enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or
inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells,
and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability
for these types of cells and discuss future directions towards in vivo applications.
We use stroboscopic quantitative phase microscopy to study cell deformation and the response to cavitation bubbles and transient shear stress resulting from laser-induced breakdown of an optically trapped nanoparticle. A bi-directional transient displacement of cytoplasm is observed during expansion and collapse of the cavitation bubble. In some cases, cell deformation is only observable at the microsecond time scale without any permanent change in cell shape or optical thickness. On a time scale of seconds, the cellular response to shear stress and cytoplasm deformation typically leads to retraction of the cellular edge most exposed to the flow, rounding of the cell body and, in some cases, loss of cellular dry mass. These results give a new insight into the cellular response to cavitation induced shear stress and related plasma membrane permeabilization. This study also demonstrates that laser-induced breakdown of a nanoparticle offers localized cavitation, which interacts with a single cell but without causing cell lysis.
Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately
injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell
biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability
of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection
have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon
effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical
transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and
throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of
femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical
reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of
DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in
the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently
trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput
microfluidic optical transfection devices.
Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of
drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low
toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput
and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and
phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic
diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the
beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply
points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in
three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of
viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan
irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.
We present a numerical technique for automatic extended focused imaging and three-dimensional analysis of
microparticle field observed in a digital holographic microscope working in transmission. We use Fourier method
for the extraction of complex amplitude from the single exposition digital holograms. We create a synthetic
extended focused image (EFI) using the focus plane determination method based on the integrated amplitude
modulus. We apply the refocusing criterion locally for each pixel, using small overlapping windows, in order to
obtain a depth map and a synthetic image in which all objects are refocused independent from their refocusing distance. The obtained synthetic EFI allows us to perform image segmentation and object detection. We improve the accuracy of vertical localization using an additional refining procedure in which each particle is treated separately. A successful application of this technique in the analysis of microgravity particle flow experiment is presented.
We present a numerical technique for refocusing and three-dimensional localization of micron-size particles observed
in a digital holographic microscope working in transmission. We use Fourier method for the extraction of
complex amplitude from the single exposition digital holograms. The three dimensional localization of objects is
performed using the focus plane determination method based on the integrated amplitude modulus. We apply
the refocusing criterion locally for each pixel, using small overlapping windows, in order to obtain a synthetic
image in which all objects are refocused independent from their refocusing distance. We perform image segmentation
and object detection using both the synthetic refocused image and the value of refocusing criterion,
which allows us to obtain a high detection efficiency with very low number of false detections. While the lateral
precision of localization is determined by the optical resolution of the setup, the vertical accuracy depends on
the parameters of the digital holographic reconstruction. We improve the accuracy of vertical localization using
an additional refining procedure in which each particle is treated separately. We analyze the robustness and
accuracy of our approach and present its successful implementation in particle flow experiments.
Bandgap guidance in photonic crystal fibers (PCF) is strongly selective spectrally and in this sense resembles guidance in fibers with Bragg gratings. The photonic band-edge shifts when the optical properties of the material in the fiber are affected by external factors such as strain, temperature, pressure, static fields, chemical vapour penetration etc. This analogy allows for the development of optical sensors with similar principle of operation as fibers with Bragg gratings, however with novel sensing characteristics. Here we study theoretically PCF filled with LC when material anisotropy plays a strong role in the creation of the bandgap.
Polarization maintaining photonic crystal fibers constitute a new class of birefringent optical fibers with strong separation of polarization modes and large possibilities of tailoring different parameters. These advantages appear to be perfect for designing optical fiber sensor, so we decided to test this type of fiber. A plane-wave method was used to numerically calculate the effective refractive indices and the field distribution of the propagation modes. The simulation results were compared with experimental measurements of the birefringence and finally the fiber was experienced as a sensor with fully automated set-up. The verification of temperature sensitivity simulations was performed too.
Holey fibers (HF) as a subgroup of photonic crystal fibers (PCF) constitute a new class of optical fibers which has revealed many interesting phenomena paving the way for a large number of novel applications either in the telecom or in the sensing domain. However, some of the applications require the use of specialty fibers with a doped core. A numerical investigation of fundamental and higher order modes propagating in doped core birefringent holey fiber is presented. The conditions for the co-existence of two competing light guiding mechanisms, their consequences on the mode propagation and the potentialities for novel applications are discussed.
We apply the fully vectorial plane wave method to calculate birefringence in photonic crystal fibers with circular and elliptical holes. Holey fibers provide the possibility of reaching extremely high modal birefringence retaining single mode guidance at the same time. Such Hi-Bi holey fibers are naturally of particular interest for sensing applications. From the technological side it is important to know which types of fiber profiles lead to the high values of modal birefringence, and the present contribution includes a comparison of selected fiber microstructures with elliptical air holes from the point of view of attainable birefringence.