Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately
injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell
biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability
of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection
have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon
effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical
transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and
throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of
femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical
reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of
DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in
the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently
trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput
microfluidic optical transfection devices.
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 year 2007 witnessed the experimental realization of extraordinary laser beams termed Airy and parabolic
beams. Surprisingly, these beams are immune to diffraction and in addition exhibit transverse acceleration while
propagating. This peculiar property of both Airy and parabolic beams facilitates the clearance of both microparticles
and cells from a region in a sample chamber through particle/cell transport along curved trajectories. We
term this concept "Optically mediated particle clearing" (OMPC) and, alternatively, "Optical redistribution"
(OR) in the presence of a microfluidic environment, where particles and cells are propelled over micrometersized
walls. Intuitively, Airy and parabolic beams act as a form of micrometer-sized "snowblower" attracting
microparticles or cells at the bottom of a sample chamber to blow them in an arc to another region of the sample.
In this work, we discuss the performance and limitations of OMPC and OR which are currently based on a single
Airy beam optionally fed by a single parabolic beam. A possible strategy to massively enhance the performance
of OMPC and OR is based on large arrays of Airy beams. We demonstrate the first experimental realization of
Efficient DNA delivery into single living cells would be a very powerful capability for cell biologists for elucidating basic cellular functions but also in other fields such as applied drug discovery and gene therapy. The ability to gently permeate the cell membrane and introduce foreign DNA with the assistance of lasers is a powerful methodology but requires exact focusing due to the required two-photon power density. Here, we demonstrate a laser-mediated delivery method of the red fluorescent protein DS-RED into Chinese hamster Ovary (CHO) cells. We used an elongated beam of light created by a Bessel beam (BB) which obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a threshold for transfection of 20%, the BB gives us transfection over twenty times the axial distance compared to the Gaussian beam of equivalent core diameter. In addition, by exploiting the BB property of reconstruction, we demonstrate successful transfection of CHO cells which involves the BB passing through an obstructive layer and re forming itself prior to reaching the cell membrane. In the light of this exciting result, one can envisage the possibility of achieving transfection through multiple cell monolayer planes and tissues using this novel light field, eliminating this way the stringent requirements for tight focusing.
Recent work has indicated the potential of light to guide the growth cones of neuronal cells using a Ti:Sapphire laser at 800 nm (Ehrlicher et al, PNAS, 2002). We have developed an optical set-up that has allowed, for the first time, the direct comparison of this process at near infrared wavelengths. A high number of growth cones were studied in order to provide a detailed statistical analysis. Actively extending growth cones of the neuroblastoma cell-line, NG108, can be guided at not only 780 nm, but also at 1064 nm. These wavelengths are an appropriate choice for guidance experiments, as wavelengths in the visible spectrum and UV are highly absorbing by cells and lead to death by phototoxicity and thermal stress. At 780 nm, 47% of actively extending growth cones were found to turn towards the focused incident light by at least 30° (n=32 growth cones). At 1064 nm, 61% of cells were successfully guided (n=31 growth cones). This suggests that the light detection mechanism within the cell is not due a single protein with a defined activity wavelength as occurs for example with the photoreceptor family of opsin proteins in the mammalian eye. We present two novel mechanisms of light induced neuronal guidance which are not related to temperature increases, or optical tweezing of the growth cone. We are also now identifying the signaling pathways that mediate this phenomenon.
The development of femtosecond (fs) lasers has continued rapidly since the demonstration of fs Ti:Sapphire systems in 1989. Recent research has yielded lasers which offer greatly enhanced performance in all areas. In this document we describe the development of femtosecond lasers with electrical to optical efficiency > 14%, pulse repetition frequencies > 4GHz and compact and stable cavities. We further outline the use of such lasers for the generation of high power visible femtosecond pulses and their application within systems environments for ultrahigh speed data communications, ultrafast optical switching and optical analogue to digital conversion. We also describe progress in the development of femtosecond lasers based on both active and passive semiconductor quantum dot components.
The introduction of naked DNA or other membrane impermeable substances into a cell (transfection) is a ubiquitous
problem in cell biology. This problem is particularly challenging when it is desired to load membrane impermeable
substances into specific cells, as most transfection technologies (such as liposomal transfection) are based on treating a
global population of cells. The technique of optical transfection, using a focused laser to open a small transient hole in
the membrane of a biological cell (photoporation) to load membrane impermeable DNA into it, allows individual cells
to be targeted for transfection, while leaving neighbouring cells unaffected. Unlike other techniques used to perform
single cell transfection, such as microinjection, optical transfection can be performed in an entirely closed system,
thereby maintaining sterility of the sample during treatment. Here, we are investigating the introduction and subsequent
expression of foreign DNA into living mammalian cells by laser-assisted photoporation with a femtosecond-pulsed
titanium sapphire laser at 800 nm, in cells that are adherent.