Focusing and Compression of Ultrashort Pulses through Scattering Media

Light scattering in inhomogeneous media induces wavefront distortions which pose an inherent limitation in many optical applications. Examples range from microscopy and nanosurgery to astronomy. In recent years, ongoing efforts have made the correction of spatial distortions possible by wavefront shaping techniques. However, when ultrashort pulses are employed scattering induces temporal distortions which hinder their use in nonlinear processes such as in multiphoton microscopy and quantum control experiments. Here we show that correction of both spatial and temporal distortions can be attained by manipulating only the spatial degrees of freedom of the incident wavefront. Moreover, by optimizing a nonlinear signal the refocused pulse can be shorter than the input pulse. We demonstrate focusing of 100fs pulses through a 1mm thick brain tissue, and 1000-fold enhancement of a localized two-photon fluorescence signal. Our results open up new possibilities for optical manipulation and nonlinear imaging in scattering media

Fluorescent carbon dioxide indicators

Over the last decade, fluorescence has become the dominant tool in biotechnology and medical imaging. These exciting advances have been underpinned by the advances in time-resolved techniques and instrumentation, probe design, chemical / biochemical sensing, coupled with our furthered knowledge in biology. Complementary volumes 9 and 10, Advanced Concepts of Fluorescence Sensing: Small Molecule Sensing and Advanced Concepts of Fluorescence Sensing: Macromolecular Sensing, aim to summarize the current state of the art in fluorescent sensing. For this reason, Drs. Geddes and Lakowicz have invited chapters, encompassing a broad range of fluorescence sensing techniques. Some chapters deal with small molecule sensors, such as for anions, cations, and CO2, while others summarize recent advances in protein-based and macromolecular sensors. The Editors have, however, not included DNA or RNA based sensing in this volume, as this were reviewed in Volume 7 and is to be the subject of a more detailed volume in the near future

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process

Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics.

We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics