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A brief review of commercial applications of femtosecond lasers in a clinical setting with emphasis on applications to corneal surgery is presented. The first clinical results of 208 procedures conducted from June to November 2000 is reported. The results show that femtosecond lasers may be safely used as keratome for use in LASIK procedures.
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Nanojoule and sub-nanojoule 80 MHz femtosecond laser pulses at 750-850 nm of a compact titanium:sapphire laser have been used for highly precise nanoprocessing of DNA as well as of intracellular and intratissue compartments. In particular, a mean power between 15 mW and 100 mW, 170 fs pulse width, submicron distance of illumination spots and microsecond beam dwell times on spots have been used for multiphoton- mediated nanoprocessing of human chromosomes, brain and ocular intrastromal tissue. By focusing the laser beam with high numerical aperture focusing optics of the laser scan system femt-O-cut and of modified multiphoton scanning microscopes to diffraction-limited spots and TW/cm2 light intensities, precise submicron holes and cuts have been processed by single spot exposure and line scans. A minimum FWHM cut size below 70 nm during the partial dissection of the human chromosome 3 was achieved. Complete chromosome dissection could be performed with FWHM cut sizes below 200 nm. Intracellular chromosome dissection was possible. Intratissue processing in depths of 50 - 100micrometers and deeper with a precision of about 1micrometers including cuts through a nuclei of a single intratissue cell without destructive photo-disruption effects to surrounding tissue layers have been demonstrated in brain and eye tissues. The femt-O-cut system includes a diagnostic system for optical tomography with submicron resolution based on multiphoton- excited autofluorescence imaging (MAI) and second harmonic generation. This system was used to localize the intracellular and intratissue targets and to control the effects of nanoprocessing. These studies show, that in contrast to conventional approaches of material processing with amplified femtosecond laser systems and (mu) J pulse energies, nanoprocessing of materials including biotissues can be performed with nJ and sub-nJ high repetition femtosecond laser pulses of turn-key compact lasers without collateral damage. Potential applications include highly precise cell and embryo surgery, gene diagnostics and gene therapy, intrastromal refractive surgery, cancer therapy and brain surgery.
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The irradiance threshold for femtosecond optical breakdown in aqueous media is approximately equals 1.0x1013W cm-2. At the breakdown threshold, a plasma with a free electron density of about 1021cm-3 is generated, and the energy density in the breakdown region is sufficiently high to cause the formation of a bubble which can be experimentally observed. We found previously that plasmas with a free electron density <1021cm-3 are formed also in a fairly large irradiance range below the breakdown threshold. The present study investigates the chemical, thermal, and thermomechanical effects produced by these low-density plasmas. We use a rate equation model considering multiphoton ionization and produced by these low-density plasmas. We use a rate equation model considering multiphoton ionization and avalanche ionization to numerically simulate the temporal evolution of the free electron density during the laser pulse for a given irradiance, and to calculate the irradiance dependence of the free-electron density and volumetric energy density reached at the end of the laser pulse. The value of the energy density created by each laser pulse is then used to calculate the temperature distribution in the focal region after application of a single laser pulse and of series of pulses. The results of the temperature calculations yield, finally, the starting point for calculations of the thermoelastic stresses that are generated during the formation of the low-density plasmas. We found that, particularly for short wavelengths, a large 'tuning range' exists for the creation of spatially extremely confined chemical, thermal and mechanical effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this irradiance range, whereas at the upper end they are mixed with thermal effects and modified by thermoelastic stresses. Above the breakdown threshold, the spatial confinement is partly destroyed by cavitation bubble formation, and the laser-induced effects become more disruptive. Our simulations revealed that the highly localized ablation of intracellular structures and intranuclear chromosome dissection recently demonstrated by other researchers are probably mediated by free-electron- induced chemical bond breaking and not related to heating or thermoelastic stresses. We conclude that low density plasmas below the optical breakdown threshold can be a versatile tool for the manipulation of transparent biological media and other transparent materials. (enabling, e.g., the generation of optical waveguides in bulk glass). Low density plasmas may, however, also be a potential hazard in multiphoton microscopy and higher harmonic imaging.
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Ultrafast lasers have become a promising tool for micromachining and extremely precise ablation of all kinds of materials. Due to the low energy threshold, thermal and mechanical side effects are limited to the bu micrometers range. The neglection of side effects enables the use of ultrashort laser pulses in a broad field of medical applications. Moreover, the interaction process based on nonlinear absorption offers the opportunity to process transparent tissue three dimensionally inside the bulk. We demonstrate the feasibility of surgical procedures in different fields of medical interest: in ophthalmology intrastromal cutting and preparing of cornael flaps for refractive surgery in living animals is presented. Besides, the very low mechanical side effects enables the use of fs- laser in otoralyngology to treat ocecular bones. Moreover, the precise cutting quality can be used in fields of cardiovascular surgery for the treatment of arteriosklerosis as well as in dentistry to remove caries from dental hard tissue.
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Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focused into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two- photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (~500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. In effect this is a power study that allows separation of different order effects. An application of ultrafast laser excitation that exploits many of the features outlined above is measurement of pH gradients in the skin. This is essential to skin barrier function and disruption of the gradient is thought to be an indicating factor in many skin diseases. A probe for which the fluorescence lifetime varies with pH is used. We thus are able to tackle problems associated with inhomogeneous labeling. We have developed a two-photon laser-scanning lifetime microscope and present pH maps of skin obtained with this instrument.
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Techniques of coherent nonlinear spectroscopy (second harmonic generation and third harmonic generation) are combined with near-field scanning optical microscopy for imaging selected chemical and physical environments in biological matter on a nanoscopic scale. Resonant enhancement of nonlinear signals is utilized as a method of producing chemically selective contrast while the order of the process provides environmental selectivity. Systems studied include natural killer cells and erythrocytes.
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The knowledge of the peculiarities of nonlinear optical phenomena in different types of tissue is important for the development of new techniques for optical biomedical diagnostics. In this work, two-photon fluorescence (TPF) and second harmonic generation (SHG) have been studied simultaneously in ordered native tissue under the picosecond laser irradiation. For excitation of TFP with signal level in order of SHG the samples of tendon tissue were colored by Rhodamine 6G. The colored samples were irradiated by the laser beam in the direction, which was perpendicular to biofibers. SHG and TPF exhibited the different polarization dependence. Magnitude of SHG signal was about 2.5 times larger in case when polarization of the laser beam was parallel to the collagen fibers than in the perpendicular case. In contrast to SHG, the TPF signal was not show the polarization dependence. Since TPF and SHG both quadratically depend on intensity, the changing of irradiation intensity due to scattering would have the same impact on these phenomena. Influence of the light scattering was revealed using the weak linear polarized beam. The strong polarization dependence of SHG can be explained by the low propagation of the linear polarized light in the ordered tissue.
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Advances in laser-scanning microscopy and the advent of confocal microscopy permitted the development of image correlation spectroscopy (ICS). ICS is an imaging analog of fluorescence correlation spectroscopy (FCS) optimized for measuring the aggregation state of fluorescently labeled macromolecules on the surface of biological cells. The ICS method entails spatial autocorrelation analysis of fluorescence fluctuations within an image sampled from an area of the sample as well as temporal autocorrelation analysis of fluorescence fluctuations through a time series of images. Together, the spatial/temporal autocorrelation analysis enables measurement of fluorophore concentration, aggregation state and transport properties. ICS was first implemented on a confocal laser-scanning microscope (CLSM) using single photon excitation. More recently we have extended the method for two-photon ICS as well as image cross-correlation spectroscopy (ICCS). ICCS allows measurement of co-localization of non-identical molecules labeled with fluorophores of different emission wavelengths. We present a variety of applications of the ICS and ICCS methods in cellular systems. We will discuss the measurement of the transport and clustering properties of membrane receptors by single photon ICS and two-photon ICCS. As well, we will describe how spatial ICS may be used to quantify the distribution of fluorescently labeled dendritic spines in neurons.
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Generating images of layered tissue structures can give valuable information to clinicians. However, the provision of accurate imaging of certain tissue structures, like teeth, in 3-dimensions is still a difficult problem. We present a method that relies on the use of pulsed Terahertz radiation to gain 3-dimensional information from teeth samples. The method makes use of Terahertz Pulse Imaging (TPI) to provide depth information. Example images are shown where structures in teeth at depth are rendered. We discuss issues that arise using this imaging method and propose ways in which it could be used in clinical practice.
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We performed surface and bulk processing experiments on different transparent materials with ultra short laser pulses. The investigations were performed mainly at 800 nm and at pulse widths ranging from 0.2 to 5 ps. We focused our attention on fluence and shot number dependencies to analyze possible incubation effects in the different materials and determine the damage threshold. In the multi- shot experiments we determined strong incubation effects which we attribute to laser-induced defect formation and accumulation. Inside the bulk we were able to generate dots and lines even in sub-micrometers sizes. The structures were analyzed by means of optical microscopy. Laser pulses at a pulse width above ca. 1 ps demonstrate strong self focusing which can be utilized for bulk and rear surface micro structuring. Below a certain pulse width other effects counteract self focusing and beam diffraction and fillamentation seem to dominate. Depending on focusing optics we observe strong differences in the possibility to process the bulk of transparent materials with fs laser pulses which we attribute to the effects in Kerr non- linearity. Also, the consequences of incubation effects on the structuring inside the bulk seem to depend strongly on the pulse width. We discuss the results based on possible technological relevance and the ablation mechanism involved.
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In this paper we demonstrate the possibility to produce single- and multimode waveguides with a well-defined number of guided modes in different doped and undoped glasses using fs laser pulses. In fused silica waveguides with damping values below 0.8 dB/cm have been realized. Moreover symmetrical beamsplitters and waveguides in different doped materials have been manufactured. Based on measurements of the generated refractive index changes calculations of the near-field intensity distribution of the guided light are performed and compared to experimental results.
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The femtosecond laser has become an important tool in the micromachining of transparent materials. In particular, focusing at high numerical aperture enables structuring the bulk of materials. At low numerical aperture and comparable energy, focused femtosecond pulses result in white light or continuum generation. It has proven difficult to damage transparent materials in the bulk at low NA. We have measured the threshold energy for continuum generation and for bulk damage in fused silica for numerical apertures between 0.01 and 0.65. The threshold for continuum generation exhibits a minimum near 0.05 NA, and increases quickly near 0.1 NA. Greater than 0.25 NA, no continuum is observed. The extent of the anti-stokes pedestal in the continuum spectrum decreases strongly as the numerical aperture is increased to 0.1, emphasizing that slow focusing is important for the broadest white light spectrum. We use a sensitive light scattering technique to detect the onset of damage. We are able to produce bulk damage at all numerical apertures studied. At high numerical aperture, the damae threshold is well below the critical power for self-focusing, which allows the breakdown intensity to be determined. Below 0.25 NA, the numerical aperture dependence suggests a possible change in damage mechanism.
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We use third harmonic generation (THG) microscopy to image waveguides and single-shot structural modifications produced in bulk glass using femtosecond laser pulses. THG microscopy reveals the internal structure of waveguides written with a femtosecond laser oscillator, and gives a three-dimensional view of the complicated morphology of the structural changes produced with single, above-threshold femtosecond pulses. We find that THG microscopy is as sensitive to refractive index change as differential interference contrast microscopy, while also offering the three-dimensional sectioning capabilities of a nonlinear microscopy technique. It is now possible to micromachine three-dimensional optical devices and to image these structures in three dimensions, all with a single femtosecond laser oscillator.
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Optical parametric chirped pulse amplification (OPCPA) is a scalable technology for ultrashort pulse amplification. Its major advantages include design simplicity, broad bandwidth, tunability, low B-integral, high contrast, and high beam quality. OPCPA is suitable both for scaling to high peak power as well as high average power. We describe the amplification of stretched 100 fs oscillator pulses in a three-stage OPCPA system pumped by a commercial, single- longitudinal-mode, Q-switched Nd:YAG laser. The stretched pulses were centered around 1054 nm with a FWHm bandwidth of 16.5 nm and had an energy of 0.5nJ. Using our OPCPA system, we obtained an amplified pulse energy of up to 31 mJ at a 10 Hz repetition rate. The overall conversion efficiency from pump to signal is 6%, which is the highest efficiency obtained with a commercial tabletop pump laser to date. The overall conversion efficiency is limited due to the finite temporal overlap of the seed (3 ns) with respect to the duration of the pump (8.5 ns). Within the temporal window of the seed pulse the pump to signal conversion efficiency exceeds 20%. Recompression of the amplified signal was demonstrated to 310 fs, limited by the aberrations initially present in the low energy seed imparted by the pulse stretcher. The maximum gain in our OPCPA system is 6x107, obtained through single passing of 40 mm of beta- barium borate. We present data on the beam quality obtained from our system (M2=1.1). This relatively simple system replaces a significantly more complex Ti:sapphire regenerative amplifier-based CPA system used in the front end of a high energy short pulse laser. Future improvement will include obtaining shorter amplified pulses and higher average power.
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Within the research project FEMTO, supported by the European Commission, a compact diode-pumped titanium:sapphire laser has been developed which matches the requirements of industrial systems, like compact dimensions and stable laser operation. To achieve this, the laser has been specially designed to be integrated directly into the machining system. For best process speed combined with optimal cutting quality, focus has been laid upon high repetition rates at moderate pulse energies. Typical average output powers are around 1.5W and repetition rates of up to 5 kHz. Accompanying to the laser development, a micro-machining system has been designed to meet the requirements of femtosecond laser micro-machining. In parallel to the machine development, machining processes have been investigated and optimized for different applications. The machining of delicate medical implants has been demonstrated as well as the machining system for general micro-machining of sensitive and delicate materials has been proven. Therefore, the developed machine offers the potential to boost the use of femtosecond lasers in industrial operation.
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We demonstrate that three-dimensional multiphonon microfabrication using photopolymerization can be accomplished using a small fraction of the output of a mode- locked Ti:sapphire laser by employing an appropriate commercially-available photoinitiator. Using this photoinitiator we have been able to develop resins with a broad range of physical and chemical characteristics for use in microfabrication. The combination of optical and chemical nonlinearity in the photopolymerization reaction allows us to make structures readily with submicron features. The use of a variable beam expander allows us to create features with a broad range of sizes using a single objective while avoiding out-of-focus polymerization.
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Biomedical Applications of Infrared Free-Electron Lasers
The W.M. Keck-Vanderbilt Free-electron Laser Center operates a reliable free-electron laser (FEL) that is used in human surgical trials, as well as in basic and applied sciences. The wavelength of the FEL is tunable from 2.1 micrometers to 9.6 micrometers , delivering above 50 mJ per macropulse with a repetition rate of 30 Hz. For soft tissue surgery, especially neurosurgery and surgery on the optic nerve, a wavelength of 6.45 micrometers has been found to ablate with little collateral damage. The free-electron laser beam is delivered to experiments approximately 2000 hours each year. The Center also supports several other tools useful for biomedical experiments: an optical parametric generator laser system with tunable wavelength similar to the free- electron laser except it has much lower average power; a Fourier transform infrared spectrometer to characterize samples; several devices for in vivo imaging including an optical coherence tomography setup, a two-photon fluorescent confocal microscope, and a cooled, integrating camera capable of imaging luciferin-luciferase reactions within the body of a mouse. The Center also houses a tunable, monochromatic x-ray source based on Compton backscattering of a laser off of a relativistic electron beam.
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The Vanderbilt Mark III FEL is a tunable source of coherent mid-infrared radiation occurring as a train of high- intensity (picosecond) micropulses with a repetition rate of 3GHz that continues for 3-5 microseconds (the macropulse). We have measured the spectral output of the Vanderbilt FEL as a function of time during the macropulse with ~10nm resolution in wavelength and ~20ns resolution in time. The measurement takes about one minute and gives a representation of the micropulse spectral width average over many macropulses. Data collected thus far indicates a surprising amount of structure produced by overlapping periods of growth, saturation, and decay within the macropulse. It is found that the central wavelength of the FEL slips over the course of the macropulse, and that the instantaneous output typically has a much smaller spectral bandwidth than the macropulse bandwidth. Thus, a user slicing portion fo the macropulse with a Pockel's Cell can obtain different central wavelengths by slicing at different times during the macropulse. The evolution of the macropulse spectrum as a function of cavity de-tuning and electron beam parameters is studied with the goal of improving the stability and spectral density of the FEL output.
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The Vanderbilt Mark III FEL is a tunable source of high- intensity coherent mid-infrared radiation occurring as a train of picosecond pulses spaced 350ps apart. The laser beam is transported to each laboratory under vacuum, but is typically transmitted through some distance of atmosphere before reaching the target. Losses due to absorption by water vapor and CO2 can be large, and since the bandwidth of the FEL is several percent of the wavelength, the spectrum can be altered by atmospheric absorptions. In order to provide an accurate representation of the laser spectrum delivered to the target, and to investigate any non-linear effects associated with transport of the FEL beam, we have recorded the spectrum of the FEL output using a vacuum spectrometer positioned after measured lengths of atmosphere. The spectrometer is equipped with a linear pyroelectric array which provides the laser spectrum for each pulse. Absorption coefficients are being measured for laboratory air, averaged over the bandwidth of the FEL. The high peak powers of this Fel have induced damage in common infrared-transparent materials; we are also measuring damage thresholds for several materials at various wavelengths.
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We report studies on the efficiency of mid-infrared laser ablation of bovine cortical bone using a free-electron laser when the ablation zone is irrigated with chemically inert and biocompatible perfluorocarbon compounds. Bovine bone samples were cut into slices with thicknesses ranging from 0.2mm to 4.0 mm. At wavelengths of 2.94, 6.1 and 6.45 micrometers the water and collagen in the bone matrix absorb the laser radiation; the perfluorocarbons transmit light at all these wavelengths, albeit with drastically varying absorption coefficients. The perfluorocarbons also dissipate heat and acoustical stress, and, under optimal conditions, prevent carbonization of the bone. The ablation efficiency - as well as plasma and bubble formation, acoustic signals and carbonization - are critically dependent on the molecular weight of the perfluorocarbon compound and its thickness. The ablation efficiency was determined as a function of wavelength, scanning speed, number of scans, and perfluorocarbon species and thickness. The laser fluence was estimated to be in the range 35 J/cm2-70j/cm2 for all wavelengths; the scanning speed was varied over the range 40micrometers /s-2960 micrometers /s. The ablation rate was estimated from scanning electron microscopy to be 0.5 mm/s. This is higher than that reported for ns Er:YAG and Q- switched CO2 lasers. The morphology of the ablation cuts at 2.94micrometers suggests a possible role for nonlinear absorption in the bone.
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We present in some detail a theoretical model that provides a dynamical account for the experimentally observed ablative properties of an FEL tuned near 6.45 microns. The model is based on thermal diffusion and chemical kinetics in a system of alternating layers of protein and saline as heated by an infrared Mark-III FEL. We compare exposure at 3.0 microns, where water is the sole absorber, to that at 6.45 microns, where both protein and water absorb. The picosecond pulses of the Mark-III superpulse are treated as a train of impulses. We consider the onset of both the helix-coil transition and chemical bond breaking in terms of the thermal, chemical, and mechanical properties of the system as well as laser wavelength and pulse structure.
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We have investigated the experimental consequences of two picosecond infrared lasers, both tuned to 6.45micrometers and focused on ocular tissue. The exposure conditions were comparable, other than pulse repetition rate, where an optical parametric oscillator/amplifier laser (OPA) system operates at a kilohertz and the Mark-III FEL at 3 gigahertz. In both cases, the peak intensity was near 2x1014 W/m2 and the total delivered energy was approximately 125 mJ. The Mark-III consistently ablates tissue, while the OPA fails to ablate or to damage corneal tissue. In particular, there is no experimental evidence for protein denaturation due to OPA irradiation. WE account for these observations in terms of a theoretical model based on thermal diffusion and threshold conditions for superheating and chemical kinetics. We comment on the relevance of tissue geometry.
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Biomedical Applications of UV Free-Electron Lasers
Recrystallization of tooth dentin by the application of mid- infrared (MIR) pulsed-laser irradiation is one candidate for a novel, non-invasive treatment for the prevention of tooth decay. Recrystallized dentin functions in a similar way to dental enamel. To recrystallize the dentin effectively and non-invasively it is essential to estimate quantitatively and qualitatively the laser parameters, such as the wavelength and the average power density, required for recrystallization. The laser-tissue interaction is initiated effectively by selective excitation of phosphate acid ions (PO4) in the dentin. Using a tunable, MIR Free Electron Laser (FEL) in the wavelength region of 8.8- 10.6micrometers , corresponding to intense absorption bands due to PO4 vibration modes, we have investigated macroscopically extent of surface modification of dentin, and we have obtained experimental results related to the ablation depth, the MIR absorption spectrum, and the elemental chemical composition. From these results, it was found that (1) the laser parameters at which efficient surface modification, without enhanced ablation effects, occurred were estimated to be approximately in the wavelength and average power density regions of ~9.4- 10.3micrometers and ~10-20 W/cm2, and that (2) in this region PO4 vibration modes with lower binding energy were preferentially excluded from the dentin.
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The first applications of a storage ring Free Electron Laser started in 1993 on the Super-ACO FEL with the study of the anisotropy decay of a coenzyme, NADH, allowing to understand the thermodynamical equilibrium of the different conformational states of the molecule and their hydrodynamical volume in solution. After this first one- color experiment using the time-resolved fluorescence technique, a transient absorption experiment was developed in which the system is excited with the UV FEL and is probed by Visible-UV absorption using synchrotron radiation. First results on the dynamical behavior were obtained for the acrinide molecule.
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We describe the commissioning of a novel two-color beamline at the Duke Free Electron Laser Laboratory, designed to perform time-resolved FTIR spectroscopy in a pump-probe scheme with sub-nanosecond resolution to measure dynamical processes with durations as long as ten nanoseconds. The UV pump pulses are produced by the tunable (193 to 700 nm) output of the OK-4 Storage-Ring FEL. The broadband, infrared probe pulses are generated as synchrotron radiation in a bending magnet downstream of the OK-4 wiggler. The repetition rate of the light source (2.79 MHz) is ideal for operating the interferometer in the rapid-scan, asynchronous sampling mode.
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The fourth generation light source (4GLS) is a new synchrotron radiation facility proposed for the United Kingdom. It is based on an energy recovery linac, and will consist of a suite of instruments providing radiation from the soft x-ray to the far infrared. In addition to undulator sources, three free electron lasers (FELs) are proposed. Two cavity-based lasers will provide infrared and vacuum-ultraviolet (VUV) radiation, and one FEL will use the self-amplification of spontaneous emission (SASE) phenomenon to produce extreme-ultraviolet (XUV) radiation. The combination of sources will provide unprecedented wavelength coverage, power, and timing structure. The 4GLS facility is expected to have great potential for many areas of research in the biomedical sciences. We discuss some potential biomedical applications of 4GLS FELs in, for example, the areas of macromolecular conformation dynamics, imaging, and radiation damage, and show where the unique properties of this combination of sources will benefit these areas of research.
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