Laser-assisted optoporation of cells and tissues – a mini-review

Laser microbeam techniques are presented, which permit the introduction of molecules or small particles into living cells. Possible mechanisms − including photochemical, photothermal and opto-mechanical interactions (ablations) − are induced by continuous wave (cw) or pulsed lasers of different wavelength, power, and mode of operation. Laser-assisted optoporation permits the uptake of fluorescent dyes as well as DNA plasmids for cell transfection, and, in addition to its broad application to cultivated cells, may have some clinical potential. © 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement


Introduction
Shortly after the invention of the laser by Theodore Maiman in 1960 scientists applied its light focusing properties to biological systems, e.g. living cells and tissues [1]. Numerous applications of the laser microbeam technique -including selective illumination of cell compartments or organelles, structural and functional studies, optical trapping and manipulation or microdissection -have been reported and summarized, e.g. in [2][3][4]. While laser microbeams are often used for measurement or imaging of biological parameters as well as for trapping or moving of cells in an optical tweezer system, the present mini-review is focused on micromanipulation or microdissection techniques for introducing molecules or small particles into a cell. Such techniques offer an alternative to injection via micro-needles, as summarized in [5,6], and appear promising where the mechanisms involved are reversible and where cell viability can be maintained.
Often molecules or small vesicles are taken up by cells due to passive diffusion through the cell membrane or by endocytosis. Another possibility is active pumping of metabolites through membrane proteins. If, however, none these mechanisms work, laser microscopy techniques may support the uptake, e.g. of membrane impermeable fluorescent dyes or DNA plasmids. In the latter case genes of a foreign organism can be introduced into a native genome in order to modify the functional or fluorescent properties of a cell. This process is called transfection and represents a main application of laser-assisted optoporation. The mechanisms involved include photochemical, photothermal and opto-mechanical interactions (ablations), as described in the following section.

Mechanisms
Interaction of laser radiation with cells or tissues may vary depending on the wavelength of irradiation as well as on the light exposure. Fairly low light doses, around 100 J/cm 2 in the visible or near ultraviolet spectral range, can induce photochemical interactions [7]. These doses increase considerably in the near infrared range, and if instead of whole cells only small areas around 1 µm 2 are irradiated, light doses up to some hundred MJ/cm 2 can be applied without photochemical cell damage [8] [16]. In furthe [18] were u w lasers indu lasers applie ell membranes to living cells laser [19]  iven in [14]: b temperature fro tion of membra occurs [15], w fter addition of medium, the tra reen fluoresce n [14]. Upon ap nd upon use o arger cell colle 7] or magneti priate heat pr -pulse (picose ablation and acromolecules epetition pulse ed for this purp r application of owed that pore gime. An impo m, offering the 0]). Often zation or they also e of gene which in a in Fig. 1 [20]. f millions e size was rtant step e prospect of high-throughput optoporation [22]. Reducing the laser pulse duration down to 25 femtoseconds increased the optoporation rate, thus confirming the importance of multiphoton effects for this mechanism [23]. Use of femtosecond lasers in combination with plasmonic gold nanoparticles further enhanced the efficiency of optoporation, since, due to an amplified localized electromagnetic field, the membrane permeability of human melanoma cells increased. Thus, a very high perforation rate of 70%, a transfection efficiency three times higher than for conventional lipofection and very low toxicity (<1%) were obtained [24].
When using a nanosecond Nd:YAG laser instead of a femtosecond laser, 25−30% of the cells were perforated at low light doses of 50 mJ/cm 2 at 532 nm, or 1 J/cm 2 at 1064 nm [25]. Targeting of diseased cells with functionalized gold nanoparticles contributed to an even more selective treatment of these cells [26]. It should be emphasized that upon application of femtosecond laser pulses, in addition to ablation, plasmonic photoionization [27] as well as thermal effects have also been described. A comparison of high repetition infrared laser pulses (1.55 µm) with cw laser irradiation of the same wavelength and average power showed that in the first case a temperature gradient was generated which was more favorable to permeabilization of cell membranes [28].

Applications
For staining cells or organelles with fluorescent dyes, their passive diffusion through the cell membrane or uptake via specific carrier systems, e.g. micelles or liposomes, is commonly used. However, cell membranes are impermeable to certain actin-staining dyes, e.g. rhodamine phalloidin [29]. In this case, laser-assisted optoporation supports the cellular uptake of these dyes, permitting visualization of the cytoskeleton. Laser-assisted cell transfection -as an alternative process to lipofection, electroporation or viral transfection (as described e.g. in [30,31]) -probably represents the broadest field of application of laser optoporation. Use of liposomes as a carrier system for DNA plasmids is reported to support their delivery to the cell nucleus and increase the transfection rate [32]. It should be mentioned that laser-assisted optoporation has often been used in combination with a laser tweezer system, where cells or particles can be trapped and moved into the focus of a (second) laser beam [3], for precise localization or interaction with microparticles [33][34][35].
A first step towards clinical application is represented by the delivery of impermeable substances into retinal explants after ultrafast laser microbeam-assisted injection [36]. Further work by the same group includes optoporation of impermeable molecules to functional cortical neurons, leading to visualization of the actin network in the growth cone, as well as delivery of impermeable molecules into targeted retinal cells in a rat's eye. This may improve visualization of the structure and function of the retina [37]. In vivo optoporation of retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles was used to label these cells specifically with fluorescent conjugates. This provides a novel approach to selectively targeting retinal cells in diseased regions while sparing neighboring healthy areas [38]. Furthermore, local ablation and injury to individual cells by a laser microbeam was used to study the calcium metabolism around epithelial wounds. Calcium influx was measured in two steps: first to the damaged cell and about 45 s later to adjacent cells, which may also have been damaged by a cavitation bubble. This demonstrated that multiple mechanisms may accompany the process of optoporation [39].
A laser microdissection and pressure catapulting technique (LMPC) has been developed [40] for the characterization of single cells and their diverse biomolecules. With LMPC, the force of focused laser light is utilized to excise selected cells or large tissue areas from object slides down to individual single cells and subcellular components like organelles or chromosomes. After microdissection, the sample is directly catapulted into an appropriate recipient vial. As this process works entirely without mechanical contact, it enables pure sample retrieval from a morphologically defined origin. LMPC has been successfully applied to isolate and catapult cells from histological tissue sections, from forensic material as well as from tough p material into the provision applications o

Conclusio
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