Knowledge of the volume of blood per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a novel method using two-photon laser scanning microscopy to obtain the local blood volume in the cortex of the anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 400 microns below the dura at the brain surface. Blood volume was calculated by normalizing the total fluorescence measured at each depth. This method, which dispenses with form recognition, is rapid and only weakly sensitive to background noise; it could be extended to measure the leakiness of the blood vessels.

Animal embryo development exhibits a complex choreography of cell movements highly regulated both in time and space. This sequence of morphogenetic movements is initiated at gastrulation and is tightly controlled by a cascade of developmental gene expression. We have recently reported that developmental gene expression can in turn be mechanically regulated by morphogenetic movements during Drosophila melanogaster early development. In order to study this phenomenon of mechanically induced gene expression, it is necessary to develop new techniques of in vivo investigation. We show that the combination of femtosecond pulse intratissue surgery and two-photon-excitation fluorescence (2PEF) microscopy is a powerful tool for (i) disrupting natural morphogenetic movements and (ii) imaging native and disrupted morphogenetic movements during Drosophila development. (i) First, non-linear-absorption-mediated photo-disruption makes it possible to perform controlled intra-vital micro-dissections resulting in the modulation of morphogenetic movements and subsequent mechano-sensitive gene expression. (ii) Second, in vivo 2PEF microscopy of transgenic GFP systems appears to be an excellent technique for long-term in vivo imaging of the complex morphogenetic movements involved in normal or perturbed Drosophila gastrulation. Together, these two techniques provide a powerful novel approach to study embryo development.

The novel compact femtosecond NIR (near infrared) laser imaging system DermaInspect was used to perform for the first time in vivo high resolution non-invasive 4D tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG). Using fast galvoscan mirrors, a time correlated single photon counting (TCSPC) module and femtosecond 80 MHz laser pulses in the spectral range of 750 nm-850 nm human skin was analyzed with subcellular spatial resolution (3D) and 250 ps temporal resolution (4D). The non-linear induced autofluorescence originates from naturally endogenous fluorophores and protein structures like NAD(P)H, flavins, phorphyrins, melanin, elastin and collagen. Collagenous structures were detected using SHG. Tissues of patients with dermatological disorders like nevi and melanoma have been investigated with a clear visualization of cells and intratissue structures. Further characterization of those components was performed by the fluorescence lifetime imaging (FLIM) and the determination of two photon excitation spectra. This method of non invasive high resolution optical biopsy provides a painless diagnostic tool for dermatological applications.

Near infrared (NIR) femtosecond laser imaging systems represent a novel and very promising diagnostic technology for non-invasive cross-sectional analysis of living biological tissues. In this study 3D multiphoton imaging with submicron resolution has been performed for non-invasive analysis of living native and tissue-engineered (TE) heart valves and blood vessels. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous structures and elastic fibers were demonstrated using multiphoton excitation at two different wavelengths. Non-invasive optical sections have been obtained without the need of staining or embedding. The quality of the resulting three-dimensional images allowed exact differentiation between collagenous structures and elastic fibers. These experimental results are very encouraging for NIR femtosecond laser scanning microscopy as a useful tool for future non-destructive monitoring and characterization of vital and intact TE cardiovascular structures.

Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H⁺ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca²⁺, and a quantitative approach to topical drug penetration.

The limit on imaging-depth in two-photon microscopy depends on parameters of the imaging system such as available power, wavelength, numerical aperture, and fluorescencecollection field-of-view and on properties of the sample such as scattering and absorption cross-sections and fluorophore distribution. These dependencies are discussed and strategies for optimizing the imaging depth are presented.

In this paper we report the use of a starch as a non-linear medium for characterising ultrashort pulses. The starch suspension in water is sandwiched between a slide holder and a cover-slip and placed within the sample plane of the nonlinear microscope. This simple arrangement enables direct measurement of the pulse where they interact with the sample.

Multiphoton excitation of photosensitizers for laser induced fluorescence diagnosis (LIFD) and photodynamic therapy (PDT) of tumors has the advantage of greater tissue penetration due to the longer wavelength of irradiation. However, multiphoton LIFD and PDT are presently not clinically applicable as there are no applicators available for the delivery of the pulsed laser radiation to the operating room. As an approach, in this contribution the beam delivery through photonic crystal fibers has been investigated. Pulses of a Ti:sapphire laser of 100 fs pulse duration and an average power of 150 mW have been transported through such a fiber of 25 m length and the resulting pulses show the absence of nonlinear contributions but still a broadening of the pulse to 2 ps due to the dispersion of the fiber. It is planned to compensate this broadening by a grating in front of the fiber. Alternatively, the transport of laser radiation of 150 fs and 100 mW through a mirror-joint-arm used for conventional CO₂ lasers has been tested showing no broadening of the laser pulses. Two-photon photodynamic activity of mTHPC-CMPEG4 shall serve as a test of the laser light transport system.

We develop a new sensor for the local in vivo measurement of optical coefficients near the surface of a tissue. To be less sensitive to the heterogeneous surface of the sample, we decided to perform space and time-resolved measurements. The sensor is a bundle of fibres. The excitation light is generated by a mode-locked Ti-Sa laser at 800nm and filtered by a 1.5nm bandwidth dielectric filter in order to limit group velocity dispersion in the monomode excitation fibre. The reflectance light is collected by gradient index fibres at 250μm and 1.3 mm from the source. The detection is performed with a Hamamatsu M5675 synchroscan streak camera. The whole system allows a time resolution of about 5ps. We made comparisons between time and space resolved Monte-Carlo numerical simulations and in vitro experimental data obtained with unskimmed UHT milk which is a known reference medium. The system does not rely on the absolute value of the reflected light intensity nor depend on the intensity ratio between different fibres since the distance between the medium and the fibres as well as the fibres tip cleanness cannot be guaranteed in vivo. As a consequence we use global characteristic of the time resolved curves such as the FWHM and their evolution with the distance from the source. The good agreement between the simulations and the experimental data lets us envisage to use numerically pre-computed tables for a real time determination of the local scattering mean free path and the anisotropy factor . We soon will be able to perform measurements with biological tissues, in vitro in a first time and in vivo in a second time.

Time-sliced and quasi continuous wave two-dimensional (2-D)transillumination imaging methods were used with independent component analysis (ICA) to generate three-dimensional (3-D)tomographic maps of absorbing and scattering inhomogeneities embedded in tissue-like turbid media. The thickness of the turbid media in both the cases was approximately 50 times the transport mean free path. The experimental arrangement for time-sliced optical imaging used 150-fs, 1 kHz repetition-rate, 800-nm light pulses from a Ti:sapphire laser system for sample illumination, and an ultrafast gated intensified camera system (UGICS) providing a minimal gate duration of 80 ps for recording 2-D images. Quasi continuous wave (CW) imaging used 784-nm CW output of a diode laser as the light source and a cooled charge coupled device (CCD) camera for recording 2-D images. Translation stages were used to scan the samples over an array of points in the x-y plane. The temporal profile of the transmitted pulse was used to extract the average optical properties of the media. An independent component separation of the signal, in conjunction with diffusive photon migration theory was used to locate the embedded inhomogeneities. An improved lateral and axial localization of the inhomogeneity over the result obtained by common photon migration reconstruction algorithm is achieved.

With the recent advent of table-top terawatt Ti:Sa laser amplifier systems, laser plasma interactions provide high-energy, femtosecond electron bunches, which might conjecture direct observation of radiation events in media of biological interest. We report on the first femtolysis studies using such laser produced relativistic electron pulses in the 2.5-15 MeV range. A real-time observation of elementary radical events is performed on water molecules and media containing an important disulfide biomolecule. The primary yield of a reducing radical produced in clusters of excitation-ionisation events (spurs) has been determined at t~3.5 10^-12 s. These data provide important information about the initial energy loss and spatial distribution of early radical events. Femtolysis studies devoted to a disulfide biomolecule is noteworthy as it is the first time that a primary ionisation event can be controlled by an ultrafast radical anion formation in the prethermal regime. This innovating domain foreshadows the development of new applications in radiobiology (microdosimetry at the nanometric scale). In the near future, electron femtolysis studies would clearly enhance the understanding of radiation-induced damages in biological confined spaces (aqueous groove of DNA and protein pockets).

The phototropins are plant blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in light, oxygen or voltage (LOV) domains. The spectroscopic properties of the LOV2 domain of phototropin 1 of Avena sativa (oat) have been investigated by means of low-temperature absorption and fluorescence spectroscopy and by time-resolved fluorescence spectroscopy. The low-temperature absorption spectrum of the LOV2 domain showed a fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket. The fluorescence quantum yield of the flavin cofactor increased from 0.13 to 0.41 upon cooling the sample from room temperature to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, allowing for an accurate positioning of the flavin triplet state in the LOV2 domain at 16900 cm^-1. Fluorescence from the cryotrapped covalent adduct state was extremely weak, with a fluorescence spectrum showing a maximum at 440 nm. Time-resolved fluorescence experiments utilizing a synchroscan streak camera revealed a singlet-excited state lifetime of the LOV2 domain of 2.4 ns. FMN dissolved in aqueous solution showed a pH-dependent lifetime ranging between 2.9 ns at pH 2.0 to 4.7 ns at pH 8.0. No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3’ terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

In this work, we have investigated, by spectrally time-resolved pump-probe spectroscopy, the excited-state dynamics of the uv mutant of the green fluorescent protein (GFPuv). The gain dynamics of GFPuv is characterized by a mono-exponential behaviour and can be described by a simple model involving a photo-conversion between two form of the GFPuv chromophore. In order to obtain more information about the role played by the interaction between the chromophore and the proteic cage, we have performed spectrally time-resolved femtosecond experiment on synthetic GFP chromophore analogue. This study allows us to evidence the importance of chromophore-proteic cage interaction in the gain dynamics. Finally we investigated the excited-state dynamics of GFPuv fused with single chain antibody fragment (scFv). The subjacent idea is to use the dynamical photo-physical properties of GFPuv fused with scFv as folding reporter. By taking two scFvs, one is a well-folded antibody and one is a misfolded antibody, we have evidenced that the observed pump probe differential transmission spectra are affected by the presence of the misfolded antibody. This result shows that the tertiary structure of the protein can be modified by the presence of a misfolded scFv linked to the GFPuv.

Tissue nonlinear spectroscopy has been investigated as potential method for the monitoring of the laser ablation process of skin tissues. Nonlinear optical phenomenon of second harmonic generation that effectively occurs when femtosecond-picosecond duration pulses of laser irradiation passed through collagen contained layer is used to monitor photothermal processes. The samples of skin tissues were irradiated by continuous wave Nd:YAG laser and then were tested by probing picosecond beam. The change in amplitude of nonlinear optical response at ablated area of sample has been revealed. As result of laser ablation in 2mm of diameter area the SHG nonlinear signal was increased approximately two times. Polarization dependence of second harmonic generation has been studied, comparing with another nonlinear process - two-photon fluorescence in samples of ordered biotissue.

An approach for the chemical imaging of molecular systems is presented, based on coherent anti-Stokes Raman scattering microscopy/spectroscopy (CARS). This technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. This offers a powerful optical tool with high sensitivity, high spatial resolution and three dimensional sectioning capability, without using fluorophores that are prone to photobleaching. CARS does not need confocal detection since the contrast mechanism results from a non-linear process (four wave mixing), that is generated only in epidetection configuration at the microscope objective focal point. We present the first results of our experiment set up on polystyrene beads.

Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.