Linear spatio-temporal characterization of a UV microscope objective for nonlinear imaging and spectroscopy by using two-dimensional spectral interferometry.

Two-dimensional Fourier transform spectral interferometry is used to characterize the spatio-temporal aberrations of a UV microscope objective. The spatial and temporal profiles of a 420 nm, 38 fs pulse at the focus of a 0.32 NA UV objective are then deduced using a wave propagation code incorporating the measured aberrations.

Simultaneous imaging of multiple focal planes using a two-photon scanning microscope.

Despite all the advances in nonlinear microscopy, all existing instruments are constrained to obtain images of one focal plane at a time. In this Letter we demonstrate a two-photon absorption fluorescence scanning microscope capable of imaging two focal planes simultaneously. This is accomplished by temporally demultiplexing the signal coming from two focal volumes at different sample depths. The scheme can be extended to three or more focal planes.

Complete characterization of a spatiotemporal pulse shaper with two-dimensional Fourier transform spectral interferometry.

Spatiotemporal pulse shaping is characterized with two-dimensional Fourier transform spectral interferometry. A deformable-mirror-based bidimensional pulse shaper is used to create simple spatiotemporal structures on a femtosecond pulse, structures that are directly calculated from the measured spatiospectral phases and intensities.

Simultaneous visualization of spatial and chromatic aberrations by two-dimensional Fourier transform spectral interferometry.

We demonstrate the use of a simple tool to simultaneously visualize and characterize chromatic and spherical aberrations that are present in multiphoton microscopy. Using two-dimensional Fourier transform spectral interferometry, we measured these aberrations, deducing in a single shot spatiotemporal effects in high-numerical-aperture objectives.

Adaptive correction of a tightly focused, high-intensity laser beam by use of a third-harmonic signal generated at an interface.

By using the third-harmonic signal generated at an air-dielectric interface, we demonstrate a novel way of correcting wavefront aberrations induced by high-numerical-aperture optics. The third harmonic is used as the input physical parameter of a genetic algorithm working in closed loop with a 37-actuator deformable mirror. This method is simple and reliable and can be used to correct aberrations of tightly focused beams, a regime where other methods have limitations. Improvement of the third-harmonic signal generated with an f/1.2 parabolic mirror by 1 order of magnitude is demonstrated.

Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis.

Growth of new micrometre sized projections called dendritic spines in neurones has been linked to the encoding of long-term memories in vertebrates. Numerous studies have been carried out at both the light and electron microscopy level to quantify dendritic spine densities in brain tissue in laboratory animals. Currently, such efforts using light microscopy have relied on manual counting of spines in confocal or two-photon optical slice images of tissue containing fluorescently labelled spines. This manual approach can be slow and tedious, especially for samples with high spine densities. We introduce an alternative way of performing spine counting that uses an applied image intensity threshold followed by spatial image correlation spectroscopy (ICS) analysis. We investigated the effect of particle sizes above the diffraction limit on the autocorrelation analysis as well as the influence of background fluorescence. Our results show that, for well labelled cerebellar tissue samples imaged with a signal-to-noise ratio of 5 or greater, ICS-based spine counts can be conducted with the same 15-20% precision as manual counting, but much more rapidly.

Nonlinear optical microscopy analysis of ultrafast phase transformation in vanadium dioxide.

The nonlinear optical properties of solid-solid phase transformation in vanadium dioxide are studied. It is found that the efficiency of the third-harmonic optical signal generated from the surface of the material increases by 1.5 orders of magnitude as a function of this phase transformation. Microscopy studies show the hysteresis of the phase transformation on a micrometer-size scale.

Femtosecond Structural Dynamics in VO2 during an Ultrafast Solid-Solid Phase Transition.

Femtosecond x-ray and visible pulses were used to probe structural and electronic dynamics during an optically driven, solid-solid phase transition in VO(2). For high interband electronic excitation (approximately 5 x 10(21) cm(-3)), a subpicosecond transformation into the high-T, rutile phase of the material is observed, simultaneous with an insulator-to-metal transition. The fast time scale observed suggests that, in this regime, the structural transition may not be thermally initiated.

Anharmonic lattice dynamics in germanium measured with ultrafast x-ray diffraction.

Damping of impulsively generated coherent acoustic oscillations in a femtosecond laser-heated thin germanium film is measured as a function of fluence by means of ultrafast x-ray diffraction. By simultaneously measuring picosecond strain dynamics in the film and in the unexcited silicon substrate, we separate anharmonic damping from acoustic transmission through the buried interface. The measured damping rate and its dependence on the calculated temperature of the thermal bath is consistent with estimated four-body, elastic dephasing times (T2) for 7-GHz longitudinal acoustic phonons in germanium.

Two-photon image correlation spectroscopy and image cross-correlation spectroscopy.

We introduce two-photon image correlation spectroscopy (ICS) using a video rate capable multiphoton microscope. We demonstrate how video rate two-photon microscopic imaging and image correlation analysis may be combined to measure molecular transport properties over ranges typical of biomolecules in membrane environments. Using two-photon ICS, we measured diffusion coefficients as large as 10(-8) cm2 s(-1) that matched theoretical predictions for samples of fluorescent microspheres suspended in aqueous sucrose solutions. We also show the sensitivity of the method for measuring microscopic flow using analogous test samples. We demonstrate explicitly the advantages of the image correlation approach for measurement of correlation functions with high signal-to-noise in relatively short time periods and discuss situations when these methods represent improvements over non-imaging fluorescence correlation spectroscopy. We present the first demonstration of two-photon image cross-correlation spectroscopy where we simultaneously excite (via two-photon absorption) non-identical fluorophores with a single pulsed laser. We also demonstrate cellular application of two-photon ICS for measurements of slow diffusion of green fluorescent protein/adhesion receptor constructs within the basal membrane of live CHO fibroblast cells.

Time-decorrelated multifocal array for multiphoton microscopy and micromachining.

We demonstrate a temporally decorrelated, multifocal multiphoton microscope. Using an etalon, we split the 800-nm light from either an ultrashort-pulsed Ti:Al (2)O (3) oscillator or a Ti:Al (2)O (3) regenerative amplifier into an array of beamlets that are delayed with respect to one another in time. The collimated beams overlap at slightly different input angles at the entrance pupil of a 1.25-numerical aperture oil-immersion objective to produce an array of foci that are temporally decorrelated at the focal plane of the objective. The temporal decorrelation eliminates any interference among the foci and permits multifocal multiphoton imaging with the resolution of single-point illumination.

Probing microscopic chemical environments with high-intensity chirped pulses.

By varying the chirp of high-intensity pulses, we can use the chirp-condition-dependent fluorescence yield to distinguish among different molecules or the same molecule in different microenvironments. As an example of the latter we show that SNAFL-2, a well-known pH-sensitive dye, shows large modulation in fluorescence yield in response to both variation in acidity and variation in chirp condition. Future application of this technique as a novel contrast mechanism within fluorescence microscopy is discussed.

Third-harmonic generation imaging of laser-induced breakdown in glass.

We present the results of three-dimensional third-harmonic generation imaging of laser-induced breakdown in glass by focused microjoule femtosecond near-IR pulses. This technique has the potential to resolve three dimensionally microstructures that result from laser-induced breakdown. As a potential optical data storage approach it is shown that the same IR laser beam can be used for writing and, at a lower power, for reading. The induced microdamage is shown to be three dimensionally confined and to depend on the write power.

Third-harmonic generation microscopy by use of a compact, femtosecond fiber laser source.

We demonstrate the first use, to our knowledge, of a compact, diode-pumped, femtosecond fiber laser for third-harmonic generation (THG) microscopy. We discuss the utility of this technique, as well as the technical issues involved in using this compact source, and demonstrate the first use, to our knowledge, of imaging by THG backlighting.

Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy.

The effects of spectral shape on two photon fluorescence excitation are investigated experimentally using an acousto-optic pulse shaper to modify femtosecond pulses from a Ti:sapphire laser. By using different spectral window shapes, we find that the measured two photon efficiency can vary by a factor of 2 for differently shaped spectra with the same full width at half maximum. We find that these effects are described well by a simple model assuming transform-limited pulses. The fact that even small changes in the spectral wings can significantly affect the efficiency of nonlinear processes has implications for biological multiphoton imaging, where it may be desirable to minimize sample exposure to radiation and maximize fluorescence or harmonic efficiency. © 1999 Society of Photo-Optical Instrumentation Engineers.

Collinear type II second-harmonic-generation frequency-resolved optical gating for use with high-numerical-aperture objectives.

Ultrashort-pulse lasers are now commonly used for multiphoton microscopy, and optimizing the performance of such systems requires careful characterization of the pulses at the tight focus of the microscope objective. We solve this problem by use of a collinear geometry in frequency-resolved optical gating that uses type II second-harmonic generation and that allows the full N.A. of the microscope objective to be used. We then demonstrate the technique by measuring the intensity and the phase of a 22-fs pulse focused by a 20x, 0.4-N.A. air objective.

Tunable short-pulse beat-wave laser source operating at 1 microm.

We have demonstrated a chirped-pulse-amplification system utilizing an air-spaced etalon inside a regenerative amplifier to produce two simultaneous 2.0-ps pulses, one centered at the gain peak of Nd:phosphate glass (1052 nm) and the other centered at the gain peak of Nd:silicate (1061 nm). Autocorrelations of the resulting beat wave demonstrate a beat frequency of 2.3 THz. We achieved wavelength tunability over a 10-nm range by electronically adjusting the etalon spacing and variable pulse width by changing the etalon rotation.

Photodisruption in the human cornea as a function of laser pulse width.

BACKGROUND: We investigated the role of laser pulse width in determining fluence thresholds and efficiency for corneal photodisruption. METHODS: A laser system that delivers a wide range of pulse energies and pulse widths was used to produce ablations at pulse widths from 100 femtoseconds (fs) to 7 nanoseconds (ns). The laser-induced breakdown fluence threshold at each pulse width was determined by monitoring individual plasma emissions. Using multiple shots, the photodisruption threshold and cutting depth at each pulse width were determined histologically. RESULTS: Corneal breakdown thresholds decreased at a faster rate from 7 ns to approximately 10 picoseconds (ps), compared to further reductions in pulse width below 10 ps, where little variation was seen. Breakdown for pulse widths below 10 ps showed little intershot variability, resulting in highly reproducible fluence thresholds. Corneal tissue examined histologically showed similar fluence dependency. CONCLUSIONS: Corneal tissue photodisruption thresholds demonstrate pulse width dependence. At pulse widths less than 10 ps and with fluences near the breakdown threshold, ablations are maximally precise and efficient. These findings suggest optimal laser parameters for corneal surgery.