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2015, Optically Induced Nanostructures
Stem cell research attracts considerable attention in developmental biology, nanobiotechnology, and medicine. Since Maksimow's landmark hypothesis on stem cells in 1909, hundreds of thousands of reports have been published in biology, medicine and biotechnology regarding stem cells, evidence of their considerable worth as a major research subject. Artificial stem cells such as 'induced pluripotent stem cells' (iPS cells), which morphologically and functionally resemble embryonic stem cells have been generated through the reprogramming of somatic cells for research purposes. Typically, optical techniques such as light microscopy in combination with fluorescent markers are applied, sometimes accompanied with fluorescence-activated cell sorting (FACS), to characterize and isolate both tissue-specific stem and iPS cells. To date, modern laserbased techniques such as nonlinear multiphoton microscopy and optical transfection using femtosecond lasers are only used in a few laboratories. The application of innovative femtosecond laser techniques in stem cell research shall be the subject of this chapter. Of particular focus is the application of femtosecond laser microscopy (i) for imaging and characterization of stem cells with high spatial sub-cellular resolution; (ii) as a nanoscalpel for optical cleaning; and (iii) for transfection and optical reprogramming of cells.
Journal of Biophotonics, 2008
Novel ultracompact multiphoton sub-20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two-photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two-photon imaging. Ultracompact low power sub-20 fs laser systems may become interesting tools for optical nanobiotechnology such as optical cleaning of stem cell clusters as well as optical transfection.
Multiphoton microscopes become important tools for noncontact sub-wavelength three-dimensional nanoprocessing of living biological specimens based on multiphoton ionization and plasma formation. Ultrashort laser pulses are required, however, dispersive effects limit the shortest pulse duration achievable at the focal plane. We report on a compact nonlinear laser scanning microscope with sub-20 femtosecond 75 MHz near infrared laser pulses for nanosurgery of human stem cells and two-photon high-resolution imaging. Single point illumination of the cell membrane was performed to induce a transient nanopore for the delivery of extracellular green fluorescent protein plasmids. Mean powers of less than 7 mW (<93 pJ) and low millisecond exposure times were sufficient to transfect human pancreatic and salivary gland stem cells. Ultracompact sub-20 femtosecond laser microscopes may become optical tools for nanobiotechnology and nanomedicine including optical stem cell manipulation.
Journal of biophotonics, 2015
Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. Illustration of the spatially shaped fem...
Journal of biomedical optics, 2015
We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.
Journal of Biomedical Optics, 2009
Modulation of the coupling of light into a waveguide via a grating, together with a novel approach to analyzing the data, is used to investigate the attachment of human embryonal carcinoma stem cells to three substrata: silica-titania ͑representative of artificial implants͒; polylysine ͑a commonly used laboratory cell culture sub-strate͒; and mucin ͑the coating of the mucosae͒. By considering both in-coupling peak width and position, the secretion of microexudate by the cells, the formation of filopodia, and the overall change in their shape ͑spread-ing͒ can be distinguished. This cannot be achieved by the conventional microscopic imaging approach. Moreover, we obtain the kinetics of these processes with excellent time resolution.
Journal of Biomedical Optics, 2012
Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima∶ 770 nm∕830 nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles.
Scientific Reports, 2015
overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a signiicant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on diferent cells can be targeted every second inside the area of the microscope ield of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the inluence of laser power is discussed, with mention on post-treatment cell survival and optoporation-eiciency rates.
Optics Express, 2004
Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.
Journal of Biomedical Optics, 2008
Laser Manipulation of Cells and Tissues ͑hereafter called Laser Manipulation͒ is a treasure for the reader. Michael Berns, who is one of the editors ͑Karl Otto Greulich is the other editor͒, became fascinated with the use of laser microbeams to manipulate cells and tissues during his doctoral research in 1964. His curiosity and interest in this field did not subside. Indeed, from his graduate work until today, he and his students and colleagues have continued to work in this area-predominately at the Beckman Laser Institute at the
In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmidcoated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.
2014
We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in labon-a-chip devices.
Lithuanian Journal of Physics, 2010
Femtosecond Laser Induced Polymerization is an attractive direct writing technique for rapid three-dimensional (3D) micro and nanofabrication in diverse applications. Recently, it has been successfully applied for 3D scaffold fabrication required in biomedicine applications. However, there are still a lot of investigations to be done before it can be used for practical applications in tissue engineering or regenerative medicine. In this work, experimental results on production of artificial polymeric scaffolds for stem cell growth are presented. Parameters (average laser power, sample scanning speed, and developing conditions) for microfabrication in biocompatible photopolymers AKRE (AKRE37) and ORMOSIL (SZ2080) are experimentally determined. 3D custom form and size artificial scaffolds were successfully microfabricated. Adult stem cell growth on them was investigated in order to test their biocompatibility. The results of myogenic stem cell culture expansion were compared to the control growth of the same cells on the scaffolds manufactured out of commonly used biocompatible photopolymers ORMOCER (Ormocore b59) and Poly-Ethylen Glycol Di-Acrylate (PEG-DA-258). Preliminary results show FLIP technique to have potential in fabrication of artificial 3D polymeric scaffolds for cell proliferation experiments. These are the first steps in transferring FLIP fabrication method from laboratory tests to flexible manufacturing of individual scaffolds out of biocompatible and biodegradable polymers.
Journal of Biomedical Optics, 2008
Human and animal stem cells ͑rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells͒ are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation ͑SHG͒ are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine ͑phosphorylated͒ dinucleotide ͓NAD͑P͒H͔ and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids. Major emission peaks at 460 and 530 nm with typical long fluorescence lifetimes ͑ 2 ͒ of 1.8 and 2.0 ns, respectively, are measured using spectral imaging and timecorrelated single photon counting. Differentiated stem cells produce the extra cellular matrix ͑ECM͒ protein collagen, detected by SHG signals at 435 nm. Multiphoton microscopes may become novel noninvasive tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation within a 3-D microenvironment.
Recently, a friend of one of these writers told her story of using one of a healthcare product to activate her stem cells as part of regenerative medicine. Regenerative medicine is a field of medicine that seeks to repair or replace damaged or diseased tissues and organs. This can be done through a variety of methods, including stem cell therapy, tissue engineering, and gene therapy. This is a short review article on this rapid field called regenerative medicine, in particular via a new method called photobiomodulation, especially by virtue of lowintensity laser pen treatment. Hopefully it will attract more research in this interesting direction, both for laser/photobiomodulation to boost plant growth as well as in healthcare (see also: Christianto & Smarandache, 2022; Christianto, 2023).
2006
Femtosecond laser pulses have the unique ability to deposit energy into a microscopic volume in the bulk of a material that is transparent to the laser wavelength without affecting the surface of the material. Here we review the use of this capability to disrupt specifically targeted structures in live cells and animals with the goal of elucidating function and modeling
Journal of Biomedicine and Biotechnology, 2010
There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications.
Journal of Biomedical Nanotechnology, 2011
In this work, focused near-infrared (NIR) femtosecond laser pulses were used to transiently perforate the cellular membrane of targeted human embryonic kidney (HEK) cells and the uptake of extrinsic molecules into the targeted cells was observed. Various cellular responses to the laser treatments were closely analyzed to optimize several experimental parameters such as laser power, exposure time and location of laser irradiation using a membrane impermeable fluorescent dye. The optimized parameters were used to investigate the entry of a plasmid DNA encoding green fluorescent protein (GFP) into the target cells. Since laser beam with higher-than-threshold energy level will disintegrate cells, we used Matlab simulations to characterize the laser irradiance and free electron distribution caused by the femtosecond-optoporation process. The simulation results showed that the free electron distribution is much narrower than the laser irradiance, which implies that the transient perforation can even be smaller than the size of the laser focal volume. Femtosecond laser-assisted optoporation when combined with lab-on-a-chip devices can be useful in single cell-based high-throughput screening.
Lasers in Medical Science
In in vitro methods and cell culture models, femtosecond (fs) laser interaction has been employed to assess its effect on the proliferation and morphology of human skin fibroblasts. We cultured a primary human skin fibroblast cell line on a glass plate, passages 17–23. The cells were irradiated with a 90-fs laser at a wavelength of 800 nm and a repetition rate of 82 MHz. The target received an average power of 320 mW for 5, 20, and 100 s, corresponding to the radiation exposures of 22.6, 90.6, and 452.9 J/cm2, respectively. Using a laser scanning microscopy technique, the photon densities were measured to be 6.4 × 1018, 2.6 × 1019, and 1.3 × 1020 photons/cm2 in a spot area of 0.07 cm2; the recorded spectra were obtained from the laser interaction after 0.00, 1.00, 25.00, and 45.00 h. The cell count and morphological changes showed that the cultured cells were affected by laser irradiation under photon stress; some fibroblasts were killed, while others were injured and survived. We d...