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.

Photoporation is a rapidly expanding technique for the introduction of macromolecules into single cells. However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm. Photoporation of 4000 Chinese Hamster ovary cells was performed, representing the largest optical transfection study reported to date. We have investigated a range of laser fluences at the cell membrane and, at 1.2 microJ/cm(2), have found an average transfection efficiency of 50 +/- 10%. Contrary to recent literature, in which 100% efficiency is claimed, our measure of efficiency accounts for all irradiated cells, including those lost as a result of laser treatment, thereby providing a true biological measure of the technique.

Non-diffracting" beams do not spread as they propagate. This property is useful in many areas. Here, the theory, generation, properties, and applications of various "non-diffracting" beams, including the Bessel beam, Mathieu beam, and Airy beam is reviewed. Applications include imaging, micromanipulation, nonlinear optics, and optical transfection."Non-diffracting" finite energy Airy beam clearing a chamber of micro-particles.

The ability to permeate selectively the cell membrane and introduce therapeutic agents is a key goal in cell biology. Optical transfection is a powerful methodology but requires exact focusing due to the required two-photon power density. The authors use a Bessel beam that obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a minimum efficiency of 20%, the Bessel beam offers transfection at axial distances 20 times greater than that of its Gaussian equivalent. Furthermore, the authors demonstrate cell transfection beyond obstacles due to the self-healing nature of the Bessel beam.

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Abstract. The biomedical sciences have benefited immensely from photonics technologies in the last 50 years. This includes the application of minute forces that enable the trapping and manipulation of cells and single molecules. In terms of the area of biophotonics, optical manipulation has made a seminal contribution to our understanding of the dynamics of single molecules and the microrheology of cells. Here we present a review of optical manipulation, emphasizing its impact on the areas of single-molecule studies and single-cell biology, and indicating some of the key experiments in the fields.

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used RT-PCR analysis to detect SARS-CoV-2 RNA on surfaces but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered, and whether virus viability is concentration dependant. Viable virus persisted for the longest time on surgical mask material and stainless steel with a 99.9% reduction in viability by 122 and 114 hours respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 hours. Viability on the bank note was reduced second fastest, with 99.9% reduction in 75 hours. RNA on all the surfaces exhibited a one log reduction in genome copy recovery over 21 days. The findings show that SARS-CoV-2 is most stable on non-porous hydrophobic surfaces. RNA is highly stable when dried on surfaces with only one log reduction in recovery over three weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within two days. Therefore, when RNA is detected on surfaces it does not directly indicate presence of viable virus even at high CT values. Importance This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface, where RT-PCR is not an appropriate technique to determine viable virus. Unless sampled immediately after contamination it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent performance. This approach can easily be integrated with advanced multimodal live-cell microscopy and micromanipulation techniques. The efficiency of this technique depends on an understanding by the user of both biology and physics. Therefore, in this protocol we discuss the subtleties that apply to both fields, including sample preparation, alignment and calibration of laser optics and their integration into a microscopy platform. The entire protocol takes ~5 d to complete, from the initial setup of the femtosecond optical transfection system to the final stage of fluorescence imaging to assay for successful expression of the gene of interest.