The challenge for successful delivery of foreign DNA into cells in vitro, a key technique in cell and molecular biology with important biomedical implications, is to improve transfection efficiency while leaving the cell's architecture intact. Here we show that a variety of mammalian cells can be directly transfected with DNA without perturbing their structure by first creating a tiny, localized perforation in the membrane using ultrashort (femtosecond), high-intensity, near-infrared laser pulses. Not only does this superior optical technique give high transfection efficiency and cell survival, but it also allows simultaneous evaluation of the integration and expression of the introduced gene.

Objective. The nucleus pulposus (NP) of the intervertebral disc develops from the notochord. Humans and other species in which notochordal cells (NCs) disappear to be replaced by chondrocyte-like mature NP cells (MNPCs) frequently develop disc degeneration, unlike other species that retain NCs. The reasons for NC disappearance are unknown. In humans, the change in cell phenotype (to MNPCs) coincides with changes that decrease nutrient supply to the avascular disc. We undertook this study to test the hypothesis that the consequent nutrient stress could be associated with NC disappearance.Methods. We measured cell densities and metabolic rates in 3-dimensional cultures of porcine NCs and bovine MNPCs, and we determined survival rates under conditions of nutrient deprivation. We used scanning electron microscopy to examine end plate porosity of discs with NCs and those with MNPCs. Nutrientmetabolite profiles and cell viability were calculated as a function of cell density and disc size in a consumption/ diffusion mathematical model.Results. NCs were more active metabolically and more susceptible to nutrient deprivation than were MNPCs. Hypoxia increased rates of glycolysis in NCs but not in MNPCs. Higher end plate porosity in discs with NCs suggested greater nutrient supply in keeping with higher nutritional demands. Mathematical simulations and experiments using an analog disc diffusion chamber indicated that a fall in nutrient concentrations resulting from increased diffusion distance during growth and/or a fall in blood supply through end plate changes could instigate NC disappearance.Conclusion. NCs demand more energy and are less resistant to nutritional stress than MNPCs, which may shed light on the fate of NCs in humans. This provides important information about prospective NC tissue engineering approaches.The intervertebral discs are cartilaginous structures interspersed between the vertebral bodies, providing flexibility to the spinal column. They consist of 3 regions: the outer annulus fibrosus (AF) surrounding the inner nucleus pulposus (NP) and a thin hyaline cartilaginous end plate lying between the disc and the adjacent vertebral bodies. The AF and NP differ in developmental origin, with the annulus arising from the mesenchyme and the nucleus from the notochord (1,2). During development the highly hydrated NP is populated by clusters of large vacuolated notochordal cells (NCs) of distinct molecular phenotype (2,3). In humans and some other species (e.g., cattle, chondrodystrophoid dogs) but not in others (e.g., rodents, pigs), NCs disappear before maturity to be replaced by chondrocyte-like cells of unknown provenance (here called mature NP cells [MNPCs]), which synthesize a more collagenous and less hydrated matrix (1,4-6).

The distribution of microfibrils was studied immunohistochemically in intervertebral discs taken from young normal human surgical cases and from the bovine tail. Co-localization of microfibrils and elastin fibres was examined by dual immunostaining of fibrillin-1 and elastin. Collagen fibre network orientation was studied by using polarized filters. A similar microfibrillar network was seen in both bovine and human discs with network organization being completely different from region to region. In the outer annulus fibrosus (OAF), abundant microfibrils organized in bundles were mainly distributed in the interterritorial matrix. In addition, the microfibril bundles were orientated parallel to each other and co-localized highly with elastin fibres. Within each lamella, co-localized microfibrils and elastin fibres were aligned in the same direction as the collagen fibres. In the interlamellar space, a dense co-localized network, staining for both microfibrils and elastin fibres, was apparent; immunostaining for both molecules was noticeably stronger than within lamellae. In the inner annulus fibrosus, the microfibrils were predominantly visible as a filamentous mesh network, both in the interterritorial matrix and also around the cells. The microfibrils in this region co-localized with elastin fibres far less than in the OAF. In nucleus pulposus, filamentous microfibrils were organized mainly around the cells where elastin fibres were hardly detected. By contrast, sparse elastin fibres, with a few of microfibrils, were visible in the interterritorial matrix . The results of this study suggest the microfibrillar network of the annulus may play a mechanical role while that around the cells of the nucleus may be more involved in regulating cell function.

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A combination of two-photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic twophoton fluorescence and immuno-staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the cell boundaries. Extensive elastin fibre networks were found in all the joint regions investigated. The elastin was found predominantly in the superficial zone (upper 50 lm) and was aligned parallel to the articular surface. Elastin was also detected in the pericellular matrix surrounding the superficial chondrocytes; however, individual fibres could not be resolved in this region. Variations in the density and organization of the fibres were observed in different regions on the joint surface.

SummaryIn this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (10 12 W cm ±2 ) by diffraction-limited focusing of the radiation of an NIR (l = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll¯uorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell±cell interactions, and gravitational biology applications.

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