Vladislav I. Shcheslavskiy

Efficient third-harmonic generation in a thin nanocrystalline film of ZnO

Nonlinear optical conversion is studied in thin films of wide-bandgap materials. Very high conversion efficiency to the third-harmonic radiation is achieved for an unamplified femtosecond Cr4+:forsterite laser in a submicron-thick film of a nanocrystalline ZnO pulsed-laser-deposited on a fused silica substrate.

High-energy self-starting femtosecond Cr 4+ :Mg 2 SiO 4 oscillator operating at a low repetition rate

We describe a self-starting high-power femtosecond laser based on the Cr(4+):Mg(2)SiO(4) crystal that produces 17-nJ pulses of 40-fs duration at 26.5-MHz repetition rate. This low repetition rate is achieved by employment of a one-to-one telescope in the cavity. The pulse energy is five times greater than with a short-resonator laser. To our knowledge, the laser produces the highest energy ever achieved from this type of laser directly from the resonator without cavity dumping or external amplification. We believe that this laser source can be used for many applications, including nonlinear optics, microscopic imaging, and micromachining of silicon and other semiconductor materials.

Nonlinear optical susceptibility measurements of solutions using third-harmonic generation on the interface

By simultaneous measurements of the third-harmonic intensity on two interfaces, it is possible to make precise and fast measurements of the third-order nonlinear optical susceptibility of solutions. We propose a simple nonlinear optical technique that eliminates the effects of both scattering and absorption, thus allowing for characterization of colloidal solutions with possible aggregation. The proposed technique is experimentally demonstrated for a variety of solutions, both transparent and scattering, and shows very good agreement with data obtained by other means.

Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping

Cellular responses to oxygen tension have been studied extensively. Oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescence sensor. The simultaneous usage of FLIM of coenzymes as NAD(P)H and FAD(+) and PLIM of oxygen sensors could provide information about correlation of metabolic pathways and oxygen tension. We investigated correlative NAD(P)H-FLIM and oxygen sensing-PLIM for simultaneously analyzing cell metabolism and oxygen tension. Cell metabolism and pO2 were observed under different hypoxic conditions in squamous carcinoma cell cultures and in complex ex vivo systems. Increased hypoxia induced an increase of the phosphorescence lifetime of Ru(BPY)3 and in most cases a decrease in the lifetime of NAD(P)H which is in agreement to the expected decrease of the protein-bound NAD(P)H during hypoxia. Oxygen was modulated directly in the mitochondrial membrane. Blocking of complex III and accumulation of oxygen could be observed by both the decrease of the phosphorescence lifetime of Ru(BPY)3 and a reduction of the lifetime of NAD(P)H which was a clear indication of acute changes in the redox state of the cells. For the first time simultaneous FLIM/PLIM has been shown to be able to visualize intracellular oxygen tension together with a change from oxidative to glycolytic phenotype.

Probing metabolic states of differentiating stem cells using two-photon FLIM

The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH is required. In our study we demonstrated, for the first time, an increased contribution of protein-bound NADPH in adipocytes that is associated with lipogenesis. The optical redox ratio FAD/NAD(P)H decreased during adipogenic differentiation, and that this was likely to be explained by the intensive biosynthesis of lipids and the enhanced NADPH production associated with this. Based on the data on the fluorescence lifetime contribution of protein-bound NAD(P)H, we registered a metabolic switch from glycolysis to oxidative phosphorylation in adipocytes.

Kilohertz gain-switched laser operation and femtosecond regenerative amplification in Cr:forsterite

We present a comprehensive study of the optimum operating regime in a gain-switched Cr:forsterite laser at a repetition frequency of 1 kHz with special attention to temperature-dependent and parasitic absorption effects and proper resonator design. On the basis of the results achieved, me demonstrate highly efficient (13% extraction efficiency) operation of a femtosecond regenerative amplifier based on Cr:forsterite and operating near room temperature with a novel BBO Pockels cell that is highly resistant to optical damage. Chirped pulse amplification raises the pulse energy to 355 /spl mu/J in /spl ap/30 cavity round trips which corresponds to an amplification factor of /spl ap/5.5/spl times/10/sup 5/. The nearly transform limited 200-/spl mu/J 135-fs compressed pulses near 1.25 /spl mu/m have a peak power of /spl ap/1.5 GW. Frequency doubling with 52% conversion efficiency in LBO produces femtosecond pulses of 104-/spl mu/J energy in the visible near 625 nm.

High volume confinement in two-photon total-internal- reflection fluorescence correlation spectroscopy

We report results on two-photon total-internal-reflection fluorescence correlation spectroscopy with radially polarized light. The combination of liquid crystal spatial light modulator, providing radial polarization, with ultrafast laser (picosecond Nd:GdVO4 laser) allowed us to take the advantage of nonlinear optical contrast mechanisms to suppress the side-lobe energy specific for radial polarization and reduce the effective excited volume twice compared to one-photon evanescent wave excitation in fluorescence correlation spectroscopy.

Third-harmonic Rayleigh scattering: theory and experiment

We present an analytical model for describing optical third-harmonic generation from a sphere that is small compared with the wavelength of light. Analysis of the problem shows that the power of the third harmonic from a sphere that is small compared with the waist size and the confocal parameter of the beam is proportional to the fourth power of a sphere’s size. Experiments with different spheres both in index-matching and non-index-matching liquids are performed and confirm theoretical predictions.

Femtosecond regenerative amplification in Cr:forsterite

We describe an oscillator-amplifier laser system for the generation of high-power femtosecond pulses near 1.25 microm based on chromium-doped forsterite. Chirped-pulse amplification at a 1-kHz repetition rate raises the pulse energy to >350 microJ. The nearly transform-limited 200-microJ, 135-fs-long recompressed pulses have a peak power of approximately 1.5 GW.

Fluorescence lifetime imaging with near-infrared dyes

Near-infrared (NIR) dyes are used as fluorescence markers in small-animal imaging and in diffuse optical tomography of the human brain. In these applications it is important to know whether the dyes bind to proteins or other tissue constituents, and whether their fluorescence lifetimes depend on the targets they are bound to. Unfortunately, neither the lasers nor the detectors of commonly used confocal and multiphoton laser scanning microscopes allow for excitation and detection of NIR fluorescence. We therefore upgraded existing confocal TCSPC FLIM systems with NIR lasers and NIR sensitive detectors. In multiphoton systems we used the Ti:Sa laser as a one-photon excitation source in combination with an NIR-sensitive detector in the confocal beam path. We tested a number of NIR dyes in biological tissue. Some of them showed clear lifetime changes depending on the tissue structures they are bound to. We therefore believe that NIR FLIM can deliver supplementary information on the tissue constitution and on local biochemical parameters.