Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

Optically-controlled platforms for transfection and single- and sub-cellular surgery

Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredictable failures or mutations in individual cells can lead to serious downstream conditions across the whole organism. The traditional tools of biochemistry struggle to observe such processes: the vast majority are based upon ensemble approaches analysing the properties of bulk populations, which means that the detail about individual constituents is lost. What are required, then, are tools with the precision and resolution to probe and dissect cells at the single-micron scale: the scale of the individual organelles and structures that control their function. In this review, we highlight the use of highly-focused laser beams to create systems providing precise control and specificity at the single cell or even single micron level. The intense focal points generated can directly interact with cells and cell membranes, which in conjunction with related modalities such as optical trapping provide a broad platform for the development of single and sub-cellular surgery approaches. These highly tuneable tools have demonstrated delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics researc

Holographic optical trapping Raman micro-spectroscopy for non-invasive measurement and manipulation of live cells

We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems

Optical Micromanipulation Techniques Combined with Microspectroscopic Methods

Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.The subject of the presented Ph.D. thesis is a combination of optical micromanipulation and microspectroscopic methods. We used laser tweezers to transport and sort various living microorganisms, such as microalgal or yeast cells. We employed Raman microspectroscopy to analyze chemical composition of individual cells and we used the information about chemical composition to automatically select the cells of interest. We combined pulsed amplitude modulation fluorescence microspectroscopy, optical micromanipulation and other techniques to map the stress response of cells to various laser wavelengths, intensities and durations of optical trapping. We fabricated microfluidic chips of various designs and we constructed Raman-tweezers sorter of micro-objects such as living cells on a microfluidic platform.

Orientation of biological cells using plane-polarized Gaussian beam optical tweezers

Optical tweezers are widely used for the manipulation of cells and their internal structures. However, the degree of manipulation possible is limited by poor control over the orientation of trapped cells. We show that it is possible to controllably align or rotate disc shaped cells - chloroplasts of Spinacia oleracea - in a plane polarised Gaussian beam trap, using optical torques resulting predominantly from circular polarisation induced in the transmitted beam by the non-spherical shape of the cells.Comment: 9 pages, 6 figure

Assembly and force measurement with SPM-like probes in holographic optical tweezers

We report a high fidelity tomographic reconstruction of the quantum state of photon pairs generated by parametric down-conversion with orbital angular momentum (OAM) entanglement. Our tomography method allows us to estimate an upper and lower bound for the entanglement between the down-converted photons. We investigate the two-dimensional state subspace defined by the OAM states ±ℓ and superpositions thereof, with ℓ=1, 2, ..., 30. We find that the reconstructed density matrix, even for OAMs up to around ℓ=20, is close to that of a maximally entangled Bell state with a fidelity in the range between F=0.979 and F=0.814. This demonstrates that, although the single count-rate diminishes with increasing ℓ, entanglement persists in a large dimensional state space