Researchers at the University of California, Irvine, US, have developed single-fiber optical tweezers for cellular micro-manipulation that demonstrated in-depth single fiber optical trapping of low- and high-index particles using micro-axicon-tip fibers. It was also demonstrated that the shape of the cone angle at the axicon's tip enabled fiber-optic trapping in the near-field. Researchers also demonstrated controlled guidance of neuronal growth, along with trapping and stretching of neurons using fiber-optic tweezers. It was also demonstrated that the cells can be stretched by the combined action of forces, such as an attractive gradient force due to fiber-optic tweezers at beam power pulling the membrane and a scattering force on the membrane. Alignment of intracellular dark material along the direction of laser beam propagation was also observed during the demonstrations.

Digital holography which enables quantitative phase evaluation during cellular microsurgery has been reported. Laser scissors have become an important tool that allows cell biologists to alter cellular structures in order to study their structure-function relationship. Digital holographic microscopy can be performed in real time, and it allows one to determine dynamic changes in the optical thickness profile of a transparent object with sub-wavelength accuracy. Quantitative phase imaging by digital holographic microscopy provided high spatial as well as temporal resolution to dynamic changes in cell thickness. A quantitative phase laser micro-irradiation system was even capable of evaluating dynamic changes in the sub-cellular structure (nucleolus) of a cell.

Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with sub-wavelength accuracy. Here, we report a quantitative phase laser microsurgery system for evaluation of cellular/ sub-cellular dynamic changes during laser micro-dissection. The proposed method takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system will permit quantitative evaluation of the damage and/or the repair of the cell or cell organelles in real time. (C) 2009 Optical Society of America

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness ('thinning') within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model.

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed.