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

Measurement of particle motion in optical tweezers embedded in a Sagnac interferometer

We have constructed a counterpropagating optical tweezers setup embedded in a Sagnac interferometer in order to increase the sensitivity of position tracking for particles in the geometrical optics regime. Enhanced position determination using a Sagnac interferometer has previously been described theoretically by Taylor et al. [Journal of Optics 13, 044014 (2011)] for Rayleigh-regime particles trapped in an antinode of a standing wave. We have extended their theory to a case of arbitrarily-sized particles trapped with orthogonally-polarized counterpropagating beams. The working distance of the setup was sufficiently long to optically induce particle oscillations orthogonally to the axis of the tweezers with an auxiliary laser beam. Using these oscillations as a reference, we have experimentally shown that Sagnac-enhanced back focal plane interferometry is capable of providing an improvement of more than 5 times in the signal-to-background ratio, corresponding to a more than 30-fold improvement of the signal-to-noise ratio. The experimental results obtained are consistent with our theoretical predictions. In the experimental setup, we used a method of optical levitator-assisted liquid droplet delivery in air based on commercial inkjet technology, with a novel method to precisely control the size of droplets.Comment: 14 pages, 8 figure

Optics and Fluid Dynamics Department annual progress report for 2002

research within three scientific programmes: (1) laser systems and optical materials, (2) optical diagnostics and information processing and (3) plasma and fluid dynamics. The department has core competences in: optical sensors, optical materials, optical storage, biophotonics, numerical modelling and information processing, non-linear dynamics and fusion plasma physics. The research is supported by several EU programmes, including EURATOM, by Danish research councils and by industry. A summary of the activities in 2002 is presented. ISBN 87-550-3197-8 (Internet

High-Speed Fluorescence Imaging and Intensity Profiling of Femtosecond-Induced Calcium Transients

We have demonstrated a combined imaging system, where the physiology of biological specimens can be imaged and profiled at 10–20 frames per second whilst undergoing femtosecond laser irradiation. Individual GH3 cells labeled with the calcium fluorophore Fluo-3 were stimulated using a counter-propagating focused femtosecond beam with respect to the imaging system. As a result of the stimulation, calcium waves can be generated in COS cells, and laser-induced calcium oscillations are initiated in the GH3 cells. Single-photon fluorescence images and intensity profiles of the targeted specimens are sampled in real-time using a modified PerkinElmer UltraView LCI microscope

An integrated optical platform for micromanipulation of cells and tissue in live animals

Thesis (Ph.D.)--Boston UniversityThe hematopoietic stem cell niche is a specialized bone marrow (BM) microenvironment where blood-forming cells reside. Interactions between these rare cells and their niche need to be studied at the single-cell level. While live animal cell tracking with optical microscopy has proven useful for this purpose, a more thorough characterization requires novel approaches. This can be accomplished by using an integrated optical platform for cell and tissue manipulations (cell transplantation and extraction) in the skull bone of live mice. The platform integrates a non-damaging laser ablation microbeam for bone removal and tissue cutting, optical tweezers for single cell trapping, and a video-rate scanning microscope. For single cell delivery, a narrow channel is ablated through bone under imaging guidance. Cells are then transferred from a micropipette into an optical trap, which brings cells into the BM through the channel. The survival and proliferation of implanted cells can be tracked in vivo by imaging. For cell extraction after laser bone thinning, different approaches can be implemented and three of them are presented

Optics and Fluid Dynamics Department annual progress report for 2001

research within three scientific programmes: (1) laser systems and optical materials, (2) optical diagnostics and information processing and (3) plasma and fluid dynamics. The department has core competences in: optical sensors, optical materials, optical storage, biooptics, numerical modelling and information processing, non-linear dynamics and fusion plasma physics. The research is supported by several EU programmes, including EURATOM, by Danish research councils and by industry. A summary of the activities in 2001 is presented. ISBN 87-550-2993-0 (Internet

Development of multiwave-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area

Three dimensional optical manipulation and structural imaging of soft materials by use of laser tweezers and multimodal nonlinear microscopy

We develop an integrated system of holographic optical trapping and multimodal nonlinear microscopy and perform simultaneous three-dimensional optical manipulation and non-invasive structural imaging of composite soft-matter systems. We combine different nonlinear microscopy techniques such as coherent anti-Stokes Raman scattering, multi-photon excitation fluorescence and multi-harmonic generation, and use them for visualization of long-range molecular order in soft materials by means of their polarized excitation and detection. The combined system enables us to accomplish both, manipulation in composite soft materials such as colloidal inclusions in liquid crystals as well as imaging of each separate constituents of the composite material in different nonlinear optical modalities. We also demonstrate optical generation and control of topological defects and simultaneous reconstruction of their three-dimensional long-range molecular orientational patterns from the nonlinear optical images

Development of multivalve-based bioprinting technology

Pluripotent stem cells (PSCs) are the most favourable sources of cells for tissue engineering applications due to their unique potency and self-renewal characteristics however they are quite fragile and can be directed to differentiate erroneously by the application of external forces. A novel multi-nozzle valve-based bioprinting platform was developed that was able to position droplets of bio-ink – such as cells in suspension – with high spatial accuracy and low impact. Volumes as low as 2 nL were successfully dispensed. Several different versions of the machine were created before the final machine was made integrating improvements and solutions to problems encountered during development. A complete evaluation of cell compatibility was carried out in order to quantify the response of cells to the bioprinting process. In the first ever study of this kind, the viability and pluripotency of human embryonic and induced pluripotent stem cells was investigated post-printing and were found to be almost completely unaffected by the bioprinting process. Many cells require a 3D culture environment in order to maintain their in vivo functions. A hybrid bioprinted-hanging-droplet technique was used to create uniform spheroid aggregates of programmable sizes from PSCs which could be used to direct PSC differentiation or as building blocks for tissue generation. Hydrogels can also be used to recreate the 3D in vivo cellular environment using the bioprinter. Alginate and hybrid polypeptide-DNA hydrogels were used, the latter for the first time with a bioprinting platform. Complex 3D structures could be created in a layer-by-layer approach with programmable heterogeneous properties throughout. Cells were added to the hydrogel precursor solution and used to bioprint 3D structures. The cells were found to be functional and highly viable while being encapsulated throughout the 3D structure of the bioprinted hydrogel which will allow the future creation of more accurate human tissue models. PSCs were successfully directed to differentiate into hepatocyte-like cells. It was shown that the bioprinting process did not interrupt or alter the pre-programmed differentiation of the cells which means that these cells can be patterned in 3D using the bioprinter while differentiating, greatly speeding up the creation of mini-liver tissue. Hepatic stellates and HUVECs were co-cultured with the hepatocyte-like cells in various ratios in an attempt to improve their hepatic function. However, no clear improvement in cytochrome P450 activity was observed indicating that further optimisation is required in this area