Near-field optics uniquely addresses problems of x, y and z resolution by spatially confining the effect of a light source to nanometric domains. The problems in using far-field optics (conventional optical imaging through a lens) to achieve nanometric spatial resolution are formidable. Near-field optics serves a bridging role in biology between optical imaging and scanned probe microscopy. The integration of near-field and scanned probe imaging with far-field optics thus holds promise for solving the so-called inverse problem of optical imaging.
The major advantages of "naked DNA gene therapy" are its simplicity and a low or negligible immune response. Gene delivery by DNA electroporation (EP) involves injection of DNA and the application of a brief electric pulse to enhance cellular permeability. Although EP is an efficient gene transduction technique in rodents, it requires much higher voltages (>500 V) in larger animals, and hence, in practice it would be hazardous for human patients, as it would cause serious tissue damage. To overcome the obstacles associated with EP-mediated gene delivery in vivo, we developed a new method of gene transduction that uses laser energy. The femtosecond infrared titanium sapphire laser beam was developed specifically for enhancing in vivo gene delivery without risks of tissue damage. System optimization revealed that injection of 10 micro g naked DNA into the tibial muscle of mice followed by application of the laser beam for 5 s, focused to 2 mm depth upon an area of 95 x 95 micro m(2), resulted in the highest intensity and duration of gene expression with no histological or biochemical evidence of muscle damage. We assessed the potential clinical application of LBGT technology by using it to transfer the murine erythropoietin (mEpo) gene into mice. LBGT-mediated mEpo gene delivery resulted in elevated (>22%) hematocrit levels that were sustained for 8 weeks. Gene expression following LBGT was detected for >100 days. Hence, LBGT is a simple, safe, effective, and reproducible method for therapeutic gene delivery with significant clinical potential.
A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.
A simple method is described for producing silver and gold nanoparticles at the tip of an atomic force microscope sensor. It is shown that these nanoparticles can enhance the nonlinear optical phenomenon of second-harmonic generation of a molecular system such as a styryl dye.
Following light absorption, the retinal chromophore of bacteriorhodospin experiences very large dipolar changes in the vertically excited state. This light-induced dipole is at least 50% larger than that of the retinal chromophore in films or in solution. We have studied the origin of the protein effect by applying second harmonic generation measurements of artificial bacteriorhodopsin pigments derived from synthetic retinal analogues, characterized by a modified polyene chain length and ring-chain conformation. The studies demonstrated a significant influence of a protein domain in the vicinity of the retinal -ionone. We suggest that tryptophane residues (specifically Trp 138 and 189) enhance the light-induced dipole in bacteriorhodopsin. The data also point to another protein domain (Trp 182) influencing the retinal chromophore. The effect of these tryptophane residues appears not only to stabilize the light-induced charge redistribution but also to enable the migration in the excited state of the positive charge into a region of the protein with a relatively low dielectric constant.
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