Non-Gaussian beam profiles such as Bessel or an- nular beams enable novel approaches to modifying materials through laser-based processing. In this review paper, proper- ties, generation methods and emerging applications for non- conventional beam shapes are discussed, including Bessel, an- nular, and vortex beams. These intensity profiles have important implications in a number of technologically relevant areas includ- ing deep-hole drilling, photopolymerization and nanopatterning, and introduce a new dimension for materials optimization and fundamental studies of laser-matter interactions.

Bessel and annular beams for materials processing

Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types—small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications a...

/pdf/intracellular-delivery-by-membrane-disruption-mechanisms-1r7lisc9lz.pdf

Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts.

Femtosecond laser ablation permits non-invasive surgeries in the bulk of a sample with submicrometer resolution. We briefly review the history of optical surgery techniques and the experimental background of femtosecond laser ablation. Next, we present several clinical applications, including dental surgery and eye surgery. We then summarize research applications, encompassing cell and tissue studies, research on C. elegans, and studies in zebrafish. We conclude by discussing future trends of femtosecond laser systems and some possible application directions.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jbio.200910053

Surgical applications of femtosecond lasers

The flat tip of an optical fiber is a unique and unconventional platform for micro and nanotechnologies The small cross-section and large aspect ratio of the fiber provide an inherently light-coupled substrate that is uniquely suited to remote, in vivo and in situ applications However, these same characteristics challenge established fabrication technologies, which are best suited to large planar substrates This review presents a broad overview of strategies for patterning the flat tip of an optical fiber Techniques discussed include self-assembly, numerous lithographies, through-fiber patterning, hybrid techniques, and strategies for mass manufacture, while the diverse applications are discussed in context throughout

The Optical Fiber Tip: An Inherently Light-Coupled Microscopic Platform for Micro- and Nanotechnologies

The plasma membrane of a eukaryotic cell is impermeable to most hydrophilic substances, yet the insertion of these materials into cells is an extremely important and universal requirement for the cell biologist. To address this need, many transfection techniques have been developed including viral, lipoplex, polyplex, capillary microinjection, gene gun and electroporation. The current discussion explores a procedure called optical injection, where a laser field transiently increases the membrane permeability to allow species to be internalized. If the internalized substance is a nucleic acid, such as DNA, RNA or small interfering RNA (siRNA), then the process is called optical transfection. This contactless, aseptic, single cell transfection method provides a key nanosurgical tool to the microscopist—the intracellular delivery of reagents and single nanoscopic objects. The experimental possibilities enabled by this technology are only beginning to be realized. A review of optical transfection is presented, along with a forecast of future applications of this rapidly developing and exciting technology.

https://royalsocietypublishing.org/doi/pdf/10.1098/rsif.2009.0463

Single cell optical transfection.

Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

/pdf/fibre-based-cellular-transfection-1a3ry1wrwf.pdf

Fibre based cellular transfection

Optical transfection is a promising technique for the delivery of foreign genetic material into cells by transiently changing the permeability of the cell membrane. Of the different optical light sources that have been used, femtosecond laser based transfection has been one of the most effective methods for optical transfection which is generally implemented using a free space bulk optical setup. In conventional optical transfection methods the foreign genetic material to be transfected is homogenously mixed in the medium. Here we report the first realization of an integrated optical transfection system which can achieve transfection along with localized drug delivery by combining a microlens fiber based optical transfection system with a micro-capillary based microfluidic system. A fiber based illumination system is also incorporated in the system in order to achieve visual identification of the cell boundaries during transfection. A novel fabrication method is devised to obtain easy and inexpensive fabrication of microlensed fibers, which can be used for femtosecond optical transfection. This fabrication method offers the flexibility to fabricate a microlens which can focus ultra-short laser pulses at a near infrared wavelength to a small focal spot (~3 µm) whilst keeping a relatively large working distance (~20 µm). The transfection efficiency of the integrated system with localized plasmid DNA delivery, is approximately 50%, and is therefore comparable to that of a standard free space transfection system. Also the use of integrated system for localized gene delivery resulted in a reduction of the required amount of DNA for transfection. The miniaturized, integrated design opens a range of exciting experimental possibilities, including the dosing of tissue slices, targeted drug delivery, and targeted gene therapy in vivo.

Integrated optical transfection system using a microlens fiber combined with microfluidic gene delivery

Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ∼6000 cores (pixels) embedded in a fiber cladding of ∼300 μm in diameter, produces an image circle (area) of ∼270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ∼72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

https://www.spiedigitallibrary.org/journals/Journal-of-Biomedical-Optics/volume-16/issue-02/028002/Optical-transfection-using-an-endoscope-like-system/10.1117/1.3541781.pdf

Optical transfection using an endoscope-like system.

We report a simple, inexpensive fabrication method to fabricate polymer microlens at the apex of optical fiber When
compared to other established protocols for fabricating microlensed fiber, this procedure allows simple and inexpensive
microlensed fiber fabrication with high reproducibility Also it is possible to tune the focal length and working distance
of the fabricated lens in a wider range by controlling the curing parameters such as curing power, beam shape of the
curing beam and curing time The novel curing procedure, where a specially structured curing laser beam of wavelength
405nm was used for curing, is the key factor for this extended flexibility Once cured, the UV curable adhesive used for
fabricating these lenses had a very good transmission in visible and near-IR region, making it ideal for communication
and biophotonics applications This fabrication technique can be used in a variety of applications owing to its ability to
achieve customized microlenses for specific applications

Nan Ma

Papers

Fibre based cellular transfection

Integrated optical transfection system using a microlens fiber combined with microfluidic gene delivery

Optical transfection using an endoscope-like system.

Fabrication of polymer microlens at the apex of optical fiber