Multiplex FISH and three-dimensional DNA imaging with near infrared femtosecond laser pulses

Abstract

We report on a novel technology for multicolor gene and chromosome detection as well as for threedimensional (3D) DNA imaging by multiphoton excitation of multiple FISH fluorophores and DNA stains. Near infrared femtosecond laser pulses at 770 nm were used to simultaneously excite the visible fluorescence of a wide range of FISH fluorophores, such as FITC, DAC, Cy3, Cy5, Cy5.5, rhodamine, spectrum aqua, spectrum green, spectrum orange, Jenfluor, and Texas red as well as of DNA/chromosome stains, for example Hoechst 33342, DAPI, SYBR green, propidium iodide, ethidium homodimer, and Giemsa. In addition to the advantage of using only one excitation wavelength for a variety of fluorophores, multiphoton excitation provided the intrinsic possibility of 3D fluorescence imaging. The technology has been used in human genetics for the diagnosis of numerical chromosome aberrations and microdeletions. In particular, multicolor 3D images of the intranuclear localization of FISH-labeled chromosome territories in interphase nuclei of amniotic fluid cells have been obtained. Using the high light penetration depth at 770 nm, optical sectioning of Hoechst 33342-labeled DNA within living culture cells and within tissue of living tumorbearing mice was performed.

Application of femtosecond‐pulsed lasers for direct optical manipulation of biological functions

“…A highly focused pulsed laser induces multiphoton absorption, which can result in multiphoton ionization in transparent materials, such as biological tissues or cells . The electron overcomes the bandgap energy and becomes a free‐ or quasi‐free electron via multiphoton absorption (Fig.…”

Mechanisms of Pulsed Laser Ablation of Biological Tissues

“…Main applications are intraocular surgery, 30,411 intrastromal corneal refractive surgery, 410,[412][413][414] and intracellular surgery. [415][416][417] In this section, we first describe the kinetics of plasma formation in biological tissues and its implications for three key parameters that characterize laser-induced breakdown: (a) the breakdown threshold, (b) plasma absorption, and (c) plasma energy density. We then discuss the thermomechanical and chemical effects induced by plasma formation and their consequences for ablation precision and efficiency.…”

“…The use of plasma-mediated ablation and disruption that are based on nonlinear absorption makes it possible to perform surgery inside of transparent biological structures. 30,[410][411][412][413][414][415][416]456 Owing to the high energy density at nanosecond and picosecond durations and the inertial confinement inside of transparent structures, the precision of plasma-mediated effects is generally compromised by cavitation effects (section X.F). Therefore, plasmamediated effects in bulk tissue are better suited for cutting and disruption than for ablation of large tissue volumes with sharply delineated boundaries.…”

“…When ultrashort laser pulses are applied at a very large numerical aperture, it is possible to perform intranuclear chromosome dissection in living cells. 415 In this procedure, the material removal per pulse is less than 0.1 µm 3 . On the other hand, standard ophthalmic applications, such as iridotomies and capsulotomies, often take advantage of the disruptive mechanical effects accompanying optical breakdown that are best achieved using nanosecond pulses.…”

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

“…Two photon excitation fluorescence (2PE) microscopy as an advanced technique offers the possibility for noninvasive, label-free high resolution imaging of living cells and deep tissues by using the fluorescence emission from the endogenous fluorescent molecules. [1][2][3] The near-infrared radiation generates 2PE signals of endogenous fluorophores, such as tryptophan, riboflavines, nicotinamides, collagen, elastin, and so on, and thus provides rich morphological and biochemical information of biological systems. [4][5][6] Less investigated and reported is the intrinsic fluorescence of hemoglobin, the main intracellular component of erythrocytes, which emits a strong Soret fluorescence with the peak at 438 nm upon two-photon excitation by femtosecond pulses in red and near-infrared region (600 to 750 nm).…”