Near-infrared Femtosecond Research Articles

Multiphoton microscopy in life sciences.

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm(-2) to GW cm(-2), which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm(-2) levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible.

Detection of luminescent single ultrasmall silicon nanoparticles using fluctuation correlation spectroscopy

We dispersed electrochemical etched Si into a colloid of ultrasmall blue luminescent nanoparticles, observable with the naked eye, in room light. We use two-photon near-infrared femtosecond excitation at 780 nm to record the fluctuating time series of the luminescence, and determine the number density, brightness, and size of diffusing fluorescent particles. The luminescence efficiency of particles is high enough such that we are able to detect a single particle, in a focal volume, of 1 pcm3. The measurements yield a particle size of 1 nm, consistent with direct imaging by transmission electron microscopy. They also yield an excitation efficiency under two-photon excitation two to threefold larger than that of fluorescein. Detection of single particles paves the way for their use as labels in biosensing applications.

Generation and Recombination of Defects in Vitreous Silica Induced by Irradiation with a Near-Infrared Femtosecond Laser

Properties of defects induced by irradiation with a near-infrared femtosecond laser beam into vitreous silica are reported. Three photoluminescence bands with photon energy of 1.9, 2.7, and 4.4 eV were observed under an excitation of 5.0 eV. An isochronal annealing experiment revealed that unrelaxed oxygen vacancies ODC(II) and interstitial oxygen were generated as dominant species during the irradiation. In the annealing, diffusion of the interstitial oxygen was thermally activated, leading to a direct reduction of oxygen-deficiency-related defects such as ODC(II) and E‘ center. The concentration of oxygen-excess-related defects such as peroxy radicals, together with nonbridging oxygen−hole centers, increased first with annealing then decreased with the exhaustion of oxygen and their precursor, the E‘ center. The recombination of oxygen vacancies with interstitial oxygen is characterized as the major process occurring during the annealing.

Switching dynamics of near-infrared vertical-cavity surface-emitting lasers by injection of near-infrared and midinfrared femtosecond pulses

We report the switch-off and recovery dynamics of a vertical-cavity surface-emitting semiconductor laser induced by injection of near-infrared or midinfrared femtosecond optical pulses. The laser is switched off by carrier-density depletion or carrier-heating effects, depending on the spectral range of the injected pulses. In both cases, the recovery of the laser output can be described by standard rate equations.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes

The influence of femtosecond near-infrared (NIR) microirradiation on cell vitality and cellular reproduction has been studied. Chinese hamster ovary cells exposed to a highly focused 150-fs scanning beam at 730, 760, and 800 nm (80 MHz, 80-mus pixel dwell time) of </=1 mW remained unaffected by the femtosecond microbeam. However, increased mean power led to impaired cell division. At >/=6 -mW mean power, cells were unable to form clones. They died or became giant cells. Complete cell destruction, including cell fragmentation, occurred at mean powers >10 mW. Cell death was accompanied by intense luminescence in the mitochondrial region. When we consider the diffraction-limited spot size in the submicrometer region, intensities and photon flux densities of 0.8-kW pulses (10-mW mean power) are of the order of terawatts per square centimeter (10(12)W/cm (2)) and 10(32) photons cm(-2) s(-1) , respectively. Extremely high fields may induce destructive intracellular plasma formation. The power limitations should be considered during NIR femtosecond microscopy of vital cells and in the design of compact NIR femtosecond solid-state lasers for two-photon microscopes.

Two-Photon near Infrared Excitation in Living Cells

Non-linear effects due to two-photon near infrared (NIR) excitation of endogenous and exogenous cellular chromophores allow novel techniques in tissue, cell and biomolecule diagnostics, as well as in intracellular micromanipulation (e.g. intracellular photochemistry). Two-photon NIR excitation may also result in cell damage effects. The high photon intensities (1024 photons cm−2 s−1) required for non-resonant two-photon excitation can be achieved by diffraction-limited focusing of continuous wave (cw) laser beams (cw microbeams) with powers in the mW range. For example, NIR traps (“laser tweezers”) used as force transducers and micromanipulation tools in cellular and molecular biology are sources of two-photon excitation. NIR traps can induce two-photon excited visible fluorescence and, in the case of &lt;800 nm-traps, UVA-like cell damage. Multimode cw microbeams may enhance non-linear effects due to longitudinal mode-beating. To perform high scan rate two-photon fluorescence imaging, the application of ultrashort laser pulses of moderate peak power but low average power (pulsed microbeams) is required. In NIR femtosecond microscopes, non-destructive imaging of two-photon excited fluorophores in various human and culture cells was demonstrated for &lt;2 mW average powers, &lt;200 mW peak powers and 400 GW cm−2 intensities (700–800 nm, ∼150 fs, ∼100 MHz). However, higher average power levels may result in failed cell reproduction and cell death due to intracellular optical breakdown. In addition, destructive transient local heating and μN force generation may occur.

Visualization of coherent nuclear motion in a membrane protein by femtosecond spectroscopy

Multicolour near-infrared femtosecond experiments of a genetically modified bacterial reaction centre show that the phase of two excited-state vibrational modes is conserved for a period of picoseconds over a large range of temperatures. The direct visualization of low-frequency nuclear vibrations in this protein, which is embedded in its natural membrane, implicates coherent nuclear motion in the primary electron transfer reaction in functional reaction centres.