INFORMACIÓN FINANCIERA AL 31 DE DICIEMBRE DEL

Fig. 73 shows the comparison of the transfection efficiency of CHO-K1 cells achieved using an axicon tipped fiber, microlens tipped fiber and the integrated system respectively, and in addition the transfection efficiency of HEK-293 treated with the integrated system. The transfection efficiency is defined as the number of cells expressing the correctly targeted red fluorescent protein 48 hours after laser treatment divided by the total number of cells that were laser treated in a particular area of interest as reported in [173]. One example of successfully transfected fluorescent cells is shown in Fig. 74. To monitor for potentially spontaneous transfected cells, each photoporated sample dish was accompanied by a control sample dish in which cells were cultured, bathed in plasmid DNA solution and then experienced the fiber presence in the absence of laser radiation. In the course of this experiment the details of the number of treated cells and the results are shown in Table 3. The number of spontaneously transfected cells varied between 0 – 2 cells for each sample dish.

Fig. 73: The transfection results of CHO-K1 and HEK-293 cells using 3 different methods. The error bar is standard error of the mean.

Table 3: Transfection results

Cell type No of Dish treated Total No. of treated cells Transfection Efficiency (±SEM) (%) Axicon tipped fiber CHO-K1 15 450 30.22 ± 5.36 Microlens tipped fiber CHO-K1 20 800 40.25 ± 3.39 Integrated system CHO-K1 15 525 45.71 ± 4.84 HEK-293 5 175 64.00 ± 4.10

The results show that the efficiency of the fiber based optical transfection technique is comparable to that of the free space transfection [174,188]. Also as shown in these results is that the microlens tipped fiber provides a higher transfection efficiency and smaller standard deviation in efficiency, than the axicon tipped fiber method. This reflects the fact that due to the longer working distance, the manipulation of a microlens tipped fiber is easier and more stable when compared to an axicon tipped fiber. During the transfection procedure, the axial focal position needed to be found only at the beginning of the procedure and then multiple cells in the same sample dish could be photoporated just by moving the fiber mount laterally. This results in less possible damage to cells and the fiber tip, a high transfection efficiency and more consistency. An integrated system providing high transfection efficiency proves that it did not have any detrimental effects to cells. The slightly higher transfection efficiency might be attributed to the higher local concentration of DNA near the transfected cells.

Fig. 74: Fluorescent microscope image of optically transfected CHO-K1 cells, incubated for 48 hours after transfection. The bright cells are transfected successfully resulting in the uptake of the plasmid and thereby expressing the mitochondrially targeted red fluorescent protein.

9.8

Conclusion

This chapter detailed a simple method to fabricate a polymer microlens at the tip of optical fiber and this technique has flexibility to allow the fabrication of customized microlenses for specific applications. The technique was used to fabricate a ‘micro-stick’ which can produce a tightly focused beam (~3 µm focal diameter) at a comparatively large working distance (~15 µm). Even though the stability requirement in this setup is high, this protocol is much easier and flexible compared to other techniques [176] to create a microlens to focus the output beam from an optical fiber. Also the optical quality of the cured UV adhesive was good, so that it was possible to use such microlensed fibers in applications which involve ultra-short laser pulses [189]. The fabricated fiber was characterized using SEM imaging, beam profiling and a ray tracing model; the parameters of the fabricated lens were estimated.

The fabrication of the collimating lens is simpler and does not require any specialized optical alignment. However it offers the flexibility to fabricate a wide range of microlenses depending on the application.

The fabrication procedure does not require any complex and expensive apparatus. Also it has been observed that this fabrication procedure is highly reproducible as all the

parameters in the fabrication process are controllable and not sensitive to the environment. The prospect of fabricating cheap polymer microlensed fiber would have potential applications in the field of communication, bio-imaging, laser nano-surgery, optical micro manipulation amongst others.

Furthermore this microstick tipped fiber was used for optical transfection of CHO- K1 and HEK-293 cells and the transfection efficiency achieved was comparable to that of conventional free space optical transfection setups and notably better than the previously reported axicon tipped fiber based optical transfection. In addition, a novel integrated system was developed to achieve localized drug (gene) delivery during transfection by combining the microlens tipped fiber with a micro-capillary system. A multimode fiber based illumination system was also combined with this integrated system to allow the efficient visual identification of the cell boundaries during optical transfection. This new technique opens up the prospect for a portable “hand-held” system that can locally deliver therapeutic agents and transfect cells within a fiber geometry placing minimal requirements upon any microscope system. This study may lead towards a wholly fiber based photoporation free system on a microscope free platform which would be compatible with endoscopic system to achieve in vivo optical transfection.

Relevant publications

• Ma N, Ashok PC, Stevenson DJ, Gunn-Moore FJ, Dholakia K (2010) Integrated optical transfection system using a microlens fiber combined with microfluidic gene delivery. Biomed Opt Express 1 (2):694-705

• Ma N, Ashok PC, Gunn-Moore FJ, Dholakia K (2010) Fabrication of polymer microlens at the apex of optical fiber. Paper presented at the Photonics 2010, Guwahati, India.

Patent

• Application: The University of St. Andrews, Ma N, Ashok PC, Stevenson DJ, Dholakia K, Gunn-Moore FJ, “ Microlensed fiber based poration” UK Patent application No. GB1009800.2

Contributions

PCA and NM came up with the idea of microlensed fabrication and design of the integrated optical transfection system. NM setup the experimental system for microlens fabrication and performed optical transfection experiments. KD and DJS gave advice regarding the design of integrated optical transfection system. FJG provided advice on the optical transfection experiments.