Aleksander Rebane

FAST CARS: Engineering a laser spectroscopic technique for rapid identification of bacterial spores

Airborne contaminants, e.g., bacterial spores, are usually analyzed by time-consuming microscopic, chemical, and biological assays. Current research into real-time laser spectroscopic detectors of such contaminants is based on e.g., resonance fluorescence. The present approach derives from recent experiments in which atoms and molecules are prepared by one (or more) coherent laser(s) and probed by another set of lasers. However, generating and using maximally coherent oscillation in macromolecules having an enormous number of degrees of freedom is challenging. In particular, the short dephasing times and rapid internal conversion rates are major obstacles. However, adiabatic fast passage techniques and the ability to generate combs of phase-coherent femtosecond pulses provide tools for the generation and utilization of maximal quantum coherence in large molecules and biopolymers. We call this technique FAST CARS (femtosecond adaptive spectroscopic techniques for coherent anti-Stokes Raman spectroscopy), and the present article proposes and analyses ways in which it could be used to rapidly identify preselected molecules in real time.

New Two-Photon Activated Photodynamic Therapy Sensitizers Induce Xenograft Tumor Regressions after Near-IR Laser Treatment through the Body of the Host Mouse

Purpose: The aim of this study was to show that novel photodynamic therapy (PDT) sensitizers can be activated by two-photon absorption in the near-IR region of the spectrum and to show, for the first time, that such activation can lead to tumor regressions at significant tissue depth. These experiments also evaluated effects of high-energy femtosecond pulsed laser irradiation on normal tissues and characterized the response of xenograft tumors to our PDT protocols. Experimental Design: Human small cell lung cancer (NCI-H69), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231) xenografts were induced in SCID mice. Irradiation of sensitized tumors was undertaken through the bodies of tumor-bearing mice to give a treatment depth of 2 cm. Posttreatment tumor regressions and histopathology were carried out to determine the nature of the response to these new PDT agents. Microarray expression profiles were conducted to assess the similarity of responses to single and two-photon activated PDT. Results: Regressions of all tumor types tested were seen. Histopathology was consistent with known PDT effects, and no, or minimal, changes were noted in irradiated normal tissues. Cluster analysis of microarray expression profiling showed reproducible changes in transcripts associated with apoptosis, stress, oxygen transport, and gene regulation. Conclusions: These new PDT sensitizers can be used at a depth of 2 cm to produce excellent xenograft regressions. The tumor response was consistent with known responses to single-photon activated PDT. Experiments in larger animals are warranted to determine the maximal achievable depth of treatment.

Dendrimer molecules with record large two-photon absorption cross section

We report what is to our knowledge a record high value for an intrinsic two-photon absorption (TPA) cross section, sigma(2) = 11 x 10(-47)> cm>(4)> s photon(-1) molecule(-1), measured with femtosecond pulses in a new dendrimer molecule comprising 29 repeat units of 4, 4(?)-bis(diphenylamino)stilbene chromophore. We measure the dependence of TPA on excitation wavelength in three consecutive generations of the dendrimer and show that the maximum sigma(2) value increases faster than the total number of stilbene chromophores. This result indicates that it is possible to obtain even larger sigma(2) values in higher generations of this dendrimer family.

Resonance enhancement of two-photon absorption in porphyrins

Abstract Two-photon absorption spectra in absolute cross-section values are presented for a number of octaethyl-, tetraphenyl-, and tetrabenzo-substituted porphyrins. In excitation spectral region of 710–810 nm, the two-photon absorption efficiency is resonantly enhanced due to nearby Q(0–0) one-photon transition. The cross-section value calculated directly from the ground- and excited-state absorption parameters in a single intermediate level approximation, agrees well with that measured in experiment.

Improved Orange and Red Ca2+ Indicators and Photophysical Considerations for Optogenetic Applications

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca(2+)) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca(2+) indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca(2+) indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca(2+) imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Picosecond time- and space-domain holography by photochemical hole burning

We demonstrate persistent storage, recall, and conjugation of picosecond light signals from various model objects, including a coin, by making use of coherent optical responses in photochemically active media. A simple linear theory of holographic storage and playback of both the spatial and the temporal behavior of the signal field is shown to describe well the experimental results obtained by utilizing octaethylporphin-doped polystyrene at 1.8 K as a spectrally selective recording material.

Enhancement of two-photon absorption in tetrapyrrolic compounds

We study the enhancement of two-photon absorption (TPA) in a series of porphyrins and tetraazaporphyrins by measuring the absolute TPA cross sections with 100-fs-duration pulses in two ranges of laser wavelengths, from 1100 to 1500 and from 700 to 800 nm. The cross section in the Q transition region is σ2∼1–10 GM (where 1 GM=10-50 cm4 s-1 photons-1), a value that is explained by partial lifting of the prohibition that is due to a parity selection rule. In the Soret transition region we find σ2 enhancement by ∼1 order of magnitude owing to the Q transition, which acts as a near-resonance intermediate state, and also owing to the presence of gerade energy levels, which we identify in this spectral region. In tetraazaporphyrins symmetrically substituted with strong electron acceptors, we find further enhancement (up to σ2∼1600 GM). As a possible application, we demonstrate for the first time to our knowledge the photosensitization of singlet-oxygen luminescence by TPA in porphyrin.

Photochemical time-domain holography of weak picosecond pulses

Abstract We show that weak picosecond optical pulse propagation through an absorbing medium with a photochemically burnedin persistent spectral hologram of a picosecond pulse train makes the sample emit coherently a replica of the pulse train applied in the burning-in cycle.

Photophysical properties and intracellular imaging of water-soluble porphyrin dimers for two-photon excited photodynamic therapy

We have investigated the photophysical properties and intracellular behaviour of a series of hydrophilic conjugated porphyrin dimers. All the dimers exhibit intense linear absorption at 650-800 nm and high singlet oxygen quantum yields (0.5-0.9 in methanol), as required for an efficient sensitiser for photodynamic therapy (PDT). They also exhibit fluorescence at 700-800 nm, with fluorescence quantum yields of up to 0.13 in methanol, and show extremely large two-photon absorption maxima of 8,000-17,000 GM in the near-IR. The dimers aggregate in aqueous solution, but aggregation is reduced by binding to bovine serum albumin (BSA), as manifested by an increase in fluorescence intensity and a sharpening in the emission bands. This process can be regarded as a model for the interaction with proteins under physiological conditions. Confocal fluorescence microscopy of live cells was used to monitor the rate of cellular uptake, intracellular localisation and photostability. Porphyrin dimers with positively charged substituents partition into cells more efficiently than the negatively charged dimers. The photostability of these dimers, in living cells, is significantly better than that of the clinical photosensitiser verteporfin. Analysis of the photophysical parameters and intracellular imaging data indicates that these dimers are promising candidates for one-photon and two-photon excited PDT.

Drastic enhancement of two-photon absorption in porphyrins associated with symmetrical electron-accepting substitution

Abstract We observe a dramatic enhancement of simultaneous two-photon absorption (TPA) in a series of substituted tetraazaporphyrins with multiple electron-accepting groups and show for the first time that the TPA cross-section increases in linear proportion with the substituents Hammett constant. The measured cross-section amounts to σ 2 =1.6×10 −47 cm 4 s/photon for octakis(4-nitrophenyl)-tetraazaporphine at 770 nm. This large value is explained by both the resonance enhancement by a strong Q x (0–0) linear transition and the presence in the same spectral region of a strong two-photon allowed gerade – gerade transition.