Gene inactivation by multiphoton-targeted photochemistry

Abstract

Multiphoton-targeted photochemistry was used to selectively inactivate the expression of genes in vertebrate cells. A membrane permeable DNA-associating vital dye, ethidium bromide monoacetate (visible wavelength single photon absorption peak at 530 nm) was used to photosensitize chromosomes in dividing cells. A 100-ps infrared laser beam operating at 1.06 microns was focused onto a selected region of a mitotic chromosome corresponding to the sites of the nucleolar (ribosomal) genes. Individual cells followed through mitosis demonstrated a reduction in the number of nucleoli formed in daughter cells that corresponded to the number of nucleolar genes sites irradiated. These results demonstrate the ability to selectively manipulate genes by using the focal point specificity characteristic of multiphoton microscopy. This technique should have wide biotechnology applications both in vitro and in vivo.

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

“…As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12,14,15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells [16][17][18][19][20][21][22], A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19,20], However, it is important to relate these physical effects to the biological response of the cells.…”

Recruitment of DNA damage recognition and repair pathway proteins following near-IR femtosecond laser irradiation of cells

“…Such events were described in laser-irradiated chromosomes more than 25 years ago using a pulsed 10-ns 532-nm Nd: YAG laser. 16,17 More recently, multiphoton-induced gene inactivation has been described following irradiation of the ribosomal gene sites on late prophase chromosomes using a 100-ps Nd: YAG laser operating at a wavelength of 1.06 m. 18 In that study, the chromosomes were sensitized with the nontoxic vital stain ethidium bromide, which had a peak absorption at 530 nm, very close to the two-photon absorption wavelength of the 1.06-m laser.…”

A polarity dependent fluorescence “switch” in live cells

“…The system described in this paper differs in concept and operation from the two-photon systems used for conventional multiphoton imaging [7], and ablation/ manipulation of subcellular structures [8,9]. The bipolar system described here is not dependent on a high photon flux generated by short-pulse lasers because the fluorescence may be induced by one photon processes or stepwise two photon processes which have much higher absorption cross-sections than the two photon virtual transition processes.…”