A. Rebane

Absolute Two-Photon Absorption Spectra and Two-Photon Brightness of Orange and Red Fluorescent Proteins

Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue two-photon microscopy. Surprisingly, little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (sigma(2)), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, sigma(2) = 315 GM, and brightness, sigma(2)phi = 130 GM, where phi is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, sigma(2) = 216 GM and sigma(2)phi = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.

A new approach to dual-color two-photon microscopy with fluorescent proteins

BackgroundTwo-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency.ResultsHere we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein.ConclusionOur method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

Slow decoherence and the radiative decay limit in rare-earth-doped crystals for coherent optical storage

We analyze the material requirements for recording, storage, and processing of optically encoded information using coherent optical transients in resonant solids. We introduce new figures of merit (FOM’s) that explicitly account for the ratio between the rate of the decoherence and the rate of the spontaneous radiative decay. Highest FOM values are achieved when the decoherence rate approaches the fundamental limit set by spontaneous emission under the condition that the total transition oscillator strength is concentrated between a single pair of energy levels. We analyze FOM’s of some of the most promising rare-earth-doped crystals at cryogenic temperatures.

Synthesis, characterization, and preclinical studies of two-photon-activated targeted PDT therapeutic triads

Photodynamic therapy (PDT) continues to evolve into a mature clinical treatment of a variety of cancer types as well as age-related macular degeneration of the eye. However, there are still aspects of PDT that need to be improved in order for greater clinical acceptance. While a number of new PDT photo-sensitizers, sometimes referred to as second- or third- generation therapeutic agents, are currently under clinical investigation, the direct treatment through the skin of subcutaneous tumors deeper than 5 mm remains problematic. Currently approved PDT porphyrin photo-sensitizers, as well as several modified porphyrins (e.g. chlorins, bacteriochlorins, etc.) that are under clinical investigation can be activated at 630-730 nm, but none above 800 nm. It would be highly desirable if new PDT paradigms could be developed that would allow photo-activation deep in the tissue transparency window in the Near-infrared (NIR) above 800 nm to reduce scattering and absorption phenomena that reduce deep tissue PDT efficacy. Rasiris and MPA Technologies have developed new porphyrins that have greatly enhanced two-photon absorption ( P A ) cross-sections and can be activated deep in the NIR (ca. 780-850 nm). These porphyrins can be incorporated into a therapeutic triad that also employs an small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR onephoton imaging agent that allows tracking the delivery of the triad to the tumor site, as well as clearance of excess triad from healthy tissue prior to the start of PDT treatment. We are currently using these new triads in efficacy studies with a breast cancer cell line that has been transfected with luciferase genes that allow implanted tumor growth and post- PDT treatment efficacy studies in SCID mouse models by following the rise and decay of the bioluminescence signal. We have also designed highly absorbing and scattering collagen breast cancer phantoms in which we have demonstrated dramatic cell kill to a depth of at least 4 cm. We have also demonstrated that at the wavelength and laser fluences used in the treatment of implanted tumors in the mouse mammary fat pads, there is little, if any, damage to the skin or internal mouse organs. In addition, we have also demonstrated that the implanted tumors can be treated to a depth of more than 1 cm by direct radiation through the dorsal side of the mouse.

Synthesis, characterization and two-photon PDT efficacy studies of triads incorporating tumor targeting and imaging components

Over the past three years we have described the rationale for using new photosensitizers (PS) with greatly enhanced multi-photon absorption. In particular, we have synthesized new porphyrin-based photosensitizers that also incorporate small molecule targeting agents that direct the ensemble to over-expressed tumor receptor sites, as well as Near-infrared imaging agents that will allow practical image-guided two-photon PDT in the tissue transparency window (750-1000 nm) at laser fluences that are harmless to surrounding healthy tissue. We have previously shown (PW2006) successful treatment of human breast cancer models (MDA-MB-231) in SCID mice, and have recently extended these studies to the treatment of both human small cell (SC) (NCI-H69) and non-small cell (NSC) (A-459) models in SCID mice. We have demonstrated that lung cancer xenografts can be successfully treated by irradiating from the side of the mouse opposite the implanted tumor, thereby passing through ca. 2 cm of mouse skin, tissue and organs before encountering the bulk tumor. These results suggest that this technology can be used to treat deep subcutaneous spontaneous tumors in larger animal models (e.g. canine). We would also emphasize that the synthetic route to these triads attaches the targeting moiety in the last step of the synthesis, and can be easily changed, thus allowing a myriad of targeting agents to be employed, opening the door to the possibility of patient-specific PDT.

Two-photon absorption spectroscopy of corroles

We report simultaneous two-photon absorption (2PA) spectra in a series of substituted corroles and related porphyrins in 800-1400 nm laser wavelength range. Compared to the porphyrins, the 2PA spectrum of corroles contains a distinct and relatively high intensity peak, sigma(2) = 60-130 GM, close to twice the wavelength of Soret maximum (800-850 nm). The increase of 2PA peak cross section is explained in terms of decreased symmetry of the contracted macro-cycle, and is most likely related to relaxing of the parity selection rules that restrict 2PA in the Soret band for more symmetrical porphyrins. We also observe that the strength of the 2PA peak in Soret region decreases with the electron-withdrawing ability (increasing Hammett constant) of the side substituents, which can be explained by assuming that the corrole core itself possesses electron-accepting ability. The peak 2PA cross sections in the Q-region are much less than in Soret-region, and can be quantitatively described within the two-level approximation taking into account permanent dipole moments in the ground and excited states.

Maximum coherence in optical transitions in rare-earth-ion-activated solids

We introduce new figures of merit (FOMs) for resonant optical materials used in recording, storage, and processing of optically encoded information using coherent optical transients. The goal is to account for maximum coherence storage time as well as for efficiency of the light matter interaction quantified using the ratio between the rate of dephasing and the rate of spontaneous radiative decay. Highest FOM values are achieved when the dephasing rate approaches the fundamental limit set by spontaneous emission under the condition that the total transition oscillator strength is concentrated between a single pair of energy levels. In this case, the information (both classical and quantum) can be transferred from the radiation field to the storage medium and back at the fastest possible rate, while the loss of optically prepared coherence is minimized. We analyze FOMs of some of the most promising rare-earth-doped crystals at cryogenic temperatures and show that the homogeneous line width may approach the radiative limit in some cases even when the peak cross section remains below the fundamental limit.

Phthalocyanine molecules with extremely strong two-photon absorption for 3D rewritable optical information storage

Phthalocyanines (Pcs) show exceptional stability against high temperatures (up to 900°C, for certain metallophthalocyanines), harsh chemical environments (strong acids and bases), γ-radiation (up to 100 MRad) and neutron radiation (up to 1019 thermal neutrons/cm2). On the other hand, Pcs exhibit a number of unique physical properties, including semi-conductivity, photoconductivity, large linear and nonlinear optical coefficients, and the ability of photo-switch between two different forms, in case of non-symmetrical metal-free Pcs. This has led to an advancement of phthalocyanine-based prototype field-effect transistors, gas- and photo-sensors, solar cells, optical power limiters, and optical memory devices (CDs). For increasing the capacity of carriers of information, it has been suggested to use the effect of simultaneous two-photon absorption (2PA), which can allow for writing and reading information in many layers, thus resulting in Terabyte (TB) disks. Our estimation of the signal-to-noise ratio shows, however, that for fast (MB/s) processing, molecular 2PA cross section must be extremely large, σ2 > 103 - 104 GM (1GM = 10-50 cm4 s), which has not been achieved yet in any photochromic material. In this paper we demonstrate, for the first time, that some specially designed non-symmetric metal-free phthahlocyanines are almost ideally suited for TB rewritable memory due to their extremely high, resonantly enhanced, 2PA cross section (~ 104 GM) in near-IR region and their intrinsic ability of reversible photo-tautomerization at lowered (~ 100 K) temperatures. We discuss how the special technical specifications, such as short pulse laser excitation and lowered working temperature, can be satisfied for space and terrestrial application.

New Organic Dendrimers with Greatly Enhanced Multi-Photon Absorption for Photonics Applications

Tree-like dendritic structures based on identical repeat units and a variety of branching patterns are an exciting new class of materials with a myriad of potential applications at the nanophotonics level. Dendrimers offer advantages over traditional linear polymers containing a mixture of chain lengths in that they are monodisperse macromolecules amenable to detailed structure property relationship studies. For the study of multi-photon absorption (MPA), dendrimers can be designed using chromophore repeat units with known photonic properties, and the resulting highly branched three-dimensional structures allow a very high packing density per unit volume, with the number of chromophore repeat units increasing exponentially with dendrimer generation.

Narrowing of the homogeneous two-photon absorption line width in two-level dipolar system

We study narrowing of nonlinear two photon absorption line shape in system with non-zero permanent dipole moment difference between the ground- and excited state. The temperature-induced broadening of one photon absorption (1PA) and two photon absorption (2PA) line shapes is modeled by subjecting the resonance transition frequency to random fluctuations of varying amplitude and by solving numerically the corresponding two-level density matrix equation of motion. We show that under conditions, when 1PA and 2PA transitions are both far from saturation, the 2PA homogeneous line width may be as much as 25% narrower than the corresponding 1PA line width. This offers novel possibilities for reducing the temperature-induced dephasing in quantum computing, quantum memory and other applications based on coherent multi-photon interactions.