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

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.

Evolving Gene Regulatory Networks with Mobile DNA Mechanisms

This paper uses a recently presented abstract, tuneable Boolean regulatory network model extended to consider aspects of mobile DNA, such as transposons. The significant role of mobile DNA in the evolution of natural systems is becoming increasingly clear. This paper shows how dynamically controlling network node connectivity and function via transposon-inspired mechanisms can be selected for in computational intelligence tasks to give improved performance. The designs of dynamical networks intended for implementation within the slime mould Physarum polycephalum and for the distributed control of a smart surface are considered.Comment: 7 pages, 8 figures. arXiv admin note: substantial text overlap with arXiv:1303.722

Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles

The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid–lipid and lipid–peptide interactions. Biomimetic giant unilamellar vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes

Creating tissue on chip constructs in microtitre plates for drug discovery

We report upon a novel coplanar dielectrophoresis (DEP) based cell patterning system for generating transferrable hepatic cell constructs, resembling a liver-lobule, in culture. The use of paper reinforced gel substrates provided sufficient strength to enable these constructs to be transfered into 96-well plates for long term functional studies, including in the future, drug development studies. Experimental results showed that hepatic cells formed DEP field-induced structures corresponding to an array of lobule-mimetic patterns. Hepatic viability was observed over a period of 3 days by the use of a fluorescent cell staining technique, whilst the liver specific functionality of albumin secretion showed a significant enhancement due to the layer patterning of cell lines (HepG2/C3A), compared to 2D patterned cells and un-patterned control. This “build and transfer” concept could, in future, also be adapted for the layer-by-layer construction of organs-on-chip in microtitre formats

Monolithic optofluidic chips: from optical manipulation of single cells to quantum sensing of fluids

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.We report on a new class of integrated optofluidic devices, fabricated by femtosecond laser micromachining. The capability to combine optical waveguides with microfluidic channels in the same glass chip provides a very powerful platform, introducing new tools in the field of optical sensing. Two recent applications that greatly benefitted from this novel technology are on-chip optical manipulation of single cells by optical forces and optical sensing of the refractive index of fluids by quantum states of light. The specific properties of robustness, alignment free and portability of these devices pave the way to the use of these advanced sensing technologies outside the lab, in a real application environment

The how and why of lncRNA function: An innate immune perspective.

Next-generation sequencing has provided a more complete picture of the composition of the human transcriptome indicating that much of the "blueprint" is a vastness of poorly understood non-protein-coding transcripts. This includes a newly identified class of genes called long noncoding RNAs (lncRNAs). The lack of sequence conservation for lncRNAs across species meant that their biological importance was initially met with some skepticism. LncRNAs mediate their functions through interactions with proteins, RNA, DNA, or a combination of these. Their functions can often be dictated by their localization, sequence, and/or secondary structure. Here we provide a review of the approaches typically adopted to study the complexity of these genes with an emphasis on recent discoveries within the innate immune field. Finally, we discuss the challenges, as well as the emergence of new technologies that will continue to move this field forward and provide greater insight into the biological importance of this class of genes. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen