We report on imaging of microcirculation by calculating the speckle variance of optical coherence tomography (OCT) structural images acquired using a Fourier domain mode-locked swept-wavelength laser. The algorithm calculates interframe speckle variance in two-dimensional and three-dimensional OCT data sets and shows little dependence to the Doppler angle ranging from 75 degrees to 90 degrees . We demonstrate in vivo detection of blood flow in vessels as small as 25 microm in diameter in a dorsal skinfold window chamber model with direct comparison with intravital fluorescence confocal microscopy. This technique can visualize vessel-size-dependent vascular shutdown and transient vascular occlusion during Visudyne photodynamic therapy and may provide opportunities for studying therapeutic effects of antivascular treatments without on exogenous contrast agent.

The spatial control of optical absorption provided by twophoton excitation (TPE) has led to tremendous advances in microscopy 1 and microfabrication 2 . Medical applications of TPE in photodynamic therapy (PDT) 3,4 have often been suggested 5-18 , but have been made impractical by the low twophoton cross-sections of photosensitiser drugs (i.e. compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise manipulation of treatment volumes in three dimensions, to a level unattainable with current techniques. Here we present a new family of PDT drugs designed for efficient TPE, and use one of them to demonstrate selective closure of blood vessels via TPE-PDT in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of clinical photosensitisers 17 . This is the first demonstration of in vivo PDT using a photosensitiser engineered for efficient two-photon excitation.Photodynamic therapy is used to treat diseases characterised by neoplastic growth including various cancers, age-related macular degeneration (AMD) and actinic keratosis 3,4 . Cell death is induced by photoexcitation of a sensitiser, generally via production of singlet oxygen. In the absence of light the photosensitiser is benign, so systemic toxicity is rare and treatment may be repeated without acquired resistance. Two-photon excitation of the photosensitiser should allow greater precision than is attainable by conventional one-photon excitation, as a consequence of the quadratic dependence of TPE on the local light intensity -the amount of TPE is inversely proportional to the fourth power of the distance from the focus. In addition, the longer wavelengths associated with TPE allow treatment deeper into tissue, by minimising absorption from endogenous chromophores.High instantaneous photon densities are essential for two-photon excitation. Early TPE-PDT studies used nanosecond lasers, but the dominant effect was photothermal damage [5][6][7] . The advent of commercial femtosecond tuneable Ti:sapphire lasers has greatly facilitated the investigation of TPE-PDT, and the limiting factor has become the availability of suitable photosensitisers. The majority of chromophores possess low two-photon cross-sections, of the order of 1-100 Goeppert-Mayer units (1 GM = 10 -50 cm 4 s photon -1 ). For example, the two FDA-approved PDT photosensitisers, verteporfin and Photofrin (cross sections 50 GM and 10 GM respectively) 17 , are unlikely to be suitable for TPE-PDT, as the high light intensities needed to achieve a therapeutic effect are close to the thresholds for photothermal or photomechanical damage 18 .Several design strategies for TPE-PDT photosensitisers have been reported recently [11][12][13][14][15][16] , but few of these compounds have yet been studied in vitro 15 , and, to date, none have progressed to in vivo testing. Porphyrin derivatives are often effective PDT agents, as exemplified ...

Abstract. We present a method for tissue fluorescence quantification in situ using a handheld fiber optic probe that measures both the fluorescence and diffuse reflectance spectra. A simplified method to decouple the fluorescence spectrum from distorting effects of the tissue optical absorption and scattering is developed, with the objective of accurately quantifying the fluorescence in absolute units. The primary motivation is measurement of 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) concentration in tissue during fluorescence-guided resection of malignant brain tumors. This technique is validated in phantoms and ex vivo mouse tissues, and tested in vivo in a rabbit brain tumor model using ALA-PpIX fluorescence contrast. C 2010 Society of Photo-Optical Instrumentation Engineers.

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various non-oncologic conditions. Singlephoton (1-γ) PDT is limited by low specificity of the photosensitizer, leading to the damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous twophoton (2-γ) excitation with ultrafast pulses of near-infrared light. Due to the non-linear interaction mechanism, 2-γ excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-γ PDT. Here, Photofrin® has been used as the photosensitizer to demonstrate proof-of-principle of 2-γ killing of vascular endothelial cells in vitro. The 2-γ absorption properties of Photofrin were investigated in the 750-900 nm excitation wavelength range. It was shown that 2-γ excitation dominates over 1-γ excitation above 800 nm. The 2-γ absorption spectrum of Photofrin in the 800 -900 nm excitation wavelength range was measured. The 2-γ cross section decreased from about 10 GM(1 GM = 10 -50 cm 4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the

CSH is associated with a significantly increased risk of infection requiring hospitalization within 1 year following cardiac implantable electronic device surgery. Strategies aimed at reducing hematomas may decrease the long-term risk of infection. (Bridge or Continue Coumadin for Device Surgery Randomized Controlled Trial [BRUISE CONTROL]; NCT00800137).

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.

14.09.12 KB. Accepted version, ok to add to spiral, embargo period as elapsed

Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.