Subsurface femtosecond tissue alteration: selectively photobleaching macular degeneration pigments in near retinal contact.

This paper uses advances in the ultrafast manipulation of light to address a general need in medicine for a clinical approach that can provide a solution to a variety of disorders requiring subsurface tissue manipulation with ultralow collateral damage. Examples are age-related macular degeneration (AMD), fungal infections, tumors surrounded by overlying tissue, cataracts, etc. Although lasers have revolutionized the use of light in clinical settings, most lasers employed in medicine cannot address such problems of depth-selective tissue manipulation. This arises from the fact that they are mostly based on one photon based laser tissue interactions that provide a cone of excitation where the energy density is sufficiently high to excite heat or fluorescence in the entire cone. Thus, it is difficult to excite a specific depth of a tissue without affecting the overlying surface. However, the advent of femtosecond (fs) lasers has caused a revolution in multiphoton microscopy (Zipfel et al. Nat. Biotechnol. 2003, 21, 1369-1377; Denk et al. Science 1990, 248, 73-76) and fabrication (Kawata et al. Nature 2001, 412, 697-698). With such lasers, the photon energy density is only high enough for multiphoton processes in the focal volume, and this opens a new direction to address subsurface tissue manipulation. Here we show in an AMD animal model, Ccr2 KO knockout mutant mice, noninvasive, selective fs two-photon photobleaching of pigments associated with AMD that accumulate under and in ultraclose proximity to the overlying retina. Pathological evidence is presented that indicates the lack of collateral damage to the overlying retina or other surrounding tissue.

D-glucose-induced second harmonic generation response in human erythrocytes.

The first experimental results of the nonresonant second harmonic generation (SHG) studies of human erythrocytes membrane exposed to various glucose concentrations in phosphate buffered saline (PBS solution) are presented in this article. It is shown that the SHG signal from the membrane can be altered as a function of glucose concentration. The link between the variation of the SHG intensity and the membrane dielectric permittivity with glucose is established both theoretically and experimentally by comparison with time domain dielectric spectroscopy (TDDS) measurement data.

Femtosecond laser: a new intradermal DNA delivery method for efficient, long-term gene expression and genetic immunization.

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.

Near-field optics: from subwavelength illumination to nanometric shadowing.

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.

Femtosecond infrared laser-an efficient and safe in vivo gene delivery system for prolonged expression.

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.