Various methods for transfecting molecules such as DNA, RNA, proteins, or drugs with high efficiency and low toxicity
have been implemented and optimized for many different cell types. These include widely used techniques such as chemical transfection
(lipid-based techniques), the use of viral vectors and electroporation. To date, laser-assisted transfection of cells has
relied on cumbersome Argon ion lasers1,2 or large, high-power lasers capable of achieving femtosecond pulses (titanium sapphire),3,4 with a few exceptions.5-8 The work reported in this article, and also recently published in Optics Express, 9 shows how the successful transfection and creation of stable cell lines is achieved using a compact, relatively inexpensive,
violet diode laser (Toptica or Nichia, at 405 nm wavelength). Turovets et al.5 use an argon fluoride excimer laser at 193 nm to transfect plant cells. Shirahata et al.6 transfect mammalian cells using a 355nm pulsed neodymium-yttrium-aluminum garnet (Nd:YAG) with pulse duration of 10ns, and
Mohanty et al.7 also use a pulsed Nd:YAG laser of wavelength 1064 nm and pulse duration of 17 ns. Sagi et al. also transfect mammalian cells using a Holmium:YAG laser (2.1 mm wavelength).8 These wavelengths were all used to create pores in the plasma membrane or to change the permeability of the plasma membrane
through a variety of effects such as heating, absorption, photochemical effects, or the creation of reactive oxygen species.
Violet diode lasers are relatively recently commercialized optical sources. They are primarily utilized for data storage but
are increasingly used in a number of roles in biophotonics, for example in confocal imaging,10 optical trapping in combination with imaging11 and, as we report here, in targeted photoporation and transfection of cells.
An exciting aspect is that the system is wholly compatible with confocal microscopy and optical tweezers, offering new directions
for automated all-optical cell selection, poration, and imaging in a single system. This is a key development in biophotonics,
and we predict that the work will be of widespread interest to researchers using violet diodes or investigating targeted transfection,
or more efficient and less toxic means of cell transfection, especially in the case of slow-growing or non-dividing cell lines.
Figure 1. Experimental Set Up
The output from either the Nichia plain (405 nm wavelength, 25 mW output power) or Toptica Photonics (CVLS-LH050-2V1, 405nm,
40mW) diode laser was directed into a home-built inverted microscope. The beam was passed through a neutral density (ND) filter
and then expanded to fill the back aperture of a x100 magnification, oil-immersion microscope objective. A charge-coupled
device (CCD) camera, coupled below the microscope objective, allowed observation of the strongly focused laser beam and the
sample dish, which contained the cells to be transfected. The complete photoporation system is outlined in Figure 1. and
its overall dimensions were 65 ×20 × 60 cm.
A beam-shutter from a single-lens reflex camera was used to provide the short exposure times required for photoporation. Cells
were positioned by translating the sample stage in x, y, and z, such that the beam focus was at exactly the same position as the cell membrane.