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The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6\~mol/l cryoprotectant (4.65, 4.65, and 3.31\~mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at -80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4\~mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-fre e tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P\~

Objective: Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use. Methods: Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9). Results: No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05). Conclusions: The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts.

Objective/Background Aim of the current study was to localize and differentiate between tumor (glioma) and healthy tissue in rat brains on a cellular level. Near-infrared multiphoton microscopy takes advantage of the simultaneous absorption of two or more photons to analyze various materials such as cell and tissue components via the observation of endogenous fluorophores such as NAD(P)H, FAD, porphyrins, melanin, elastin, and collagen, with a very high resolution, without inducing the problems of photo-bleaching on out-of-focus areas. Methods In vitro and in vivo studies on healthy rat brains as well as C6 glioma cell line allografts have been performed. Near-infrared laser pulses (??=?6901060?nm, t similar to 140?fs) generated by an ultrafast Ti:Sapphire tunable laser system (Chameleon, Coherent GmbH, Santa Clara, CA) were coupled into a laser scanning microscope (LSM 510 META, Carl Zeiss, Germany) to observe high quality images. Results Several image acquisitions have been performed by varying the zoom scale of the multiphoton microscope, image acquisition time and the wavelength (765, 840?nm) to detect various tissue components. With a penetration depth of similar to 200?mu m in vitro and about 3060?mu m in vivo into the brain tissue it was possible to differentiate between tumor and healthy brain tissue even through thin layers of blood. Conclusion Near-infrared multiphoton microscopy allows the observation and possibly differentiation between tumor (glioma) and healthy tissue in rat brains on a cellular level. Our findings suggest that a further miniaturization of this technology might be very useful for scientific and clinical applications in neurosurgery.

Purpose. To investigate femtosecond laser-assisted nanosurgery of the anterior lens capsule in a prospective in vitro study. Methods. Eight anterior lens capsules obtained during conventional phaco surgery were irradiated with a non amplified 80-MHz near-infrared 800-nm titanium:sapphire femtosecond laser. Line intratissue laser cuts were examined by femtosecond multiphoton laser scanning microscopy (MLSM) and transmission electron microscopy (TEM). Results. Speed parameters of the laser beam, laser ablation time, and pulse power determined the width of the lesions, which ranged from 220±40 nm (SD) to 1.49±0.15 m. Both MLSM and TEM revealed minimal collateral alterations in the tissue surrounding the laser cuts. Conclusions. Non amplified near-infrared femtosecond laser pulses at low pulse energies may be a promising strategy for precise noncontact nanosurgery of the anterior lens capsule with minimal collateral damage to surrounding tissue. High-resolution MLSM offers 3-dimensional, noninvasive, nondestructive imaging at submicrometer resolution within seconds before and after ablation.

Transplantation of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. In previous studies we demonstrated that conventional frozen cryopreservation (FC) is accompanied by serious alterations of extracellular matrix (ECM) structures. As the main culprit of the observed damages ice crystal formation was identified. Objective of this study was the application principles of cryoprotection as observed in nature, occurring in animals or plants, for ice-free cryopreservation (IFC) of heart valves. Using IFC, valves were processed and stored above the glass transition temperature of the cryoprotectant formulation (-124 degrees C) at -80 degrees C to avoid any ice formation, tissue-glass cracking and preserving ECM. After implantation in the orthotopic pulmonary position in sheep, we demonstrate that IFC resulted in cell free matrices, while maintaining crucial ECM-components such as elastin and collagen, translating into superior hemodynamics. In contrast, we reveal that FC valves showed ECM damage that was not restored in vivo, and T-cell inflammation of the stroma with significant leaflet thickening. Compared to currently applied FC practice IFC also reduced infrastructural needs for preservation, storage and shipping. These results have important implications for clinical valve transplantation including the promise of better long-term function and lower costs.

Specific transport of anti-cancer drugs into tumor cells may result in increased therapeutic efficacy and decreased adverse events. Expression of alpha v beta 3 integrin is enhanced in various types of cancer and monoclonal antibodies (mAbs) directed against alpha v beta 3 integrins hold promise for anti-cancer therapy. DI17E6 is a monoclonal antibody directed against alpha v integrins that inhibits growth of melanomas in vitro and in vivo and inhibits angiogenesis due to interference with alpha v beta 3 integrins. Here, DI17E6 was covalently coupled to human serum albumin nanoparticles. Resulting nanoparticles specifically targeted alpha v beta 3 integrin positive melanoma cells. Moreover, doxorubicin loaded DI17E6 nanoparticles showed increased cytotoxic activity in alpha v beta 3-positive melanoma cells than the free drug. Therefore, DI17E6-coupled human serum albumin nanoparticles represent a potential delivery system for targeted drug transport into alpha v beta 3-positive cells.

Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.

Purpose: To investigate retinal imaging and ablation using femtosecond laser pulses. Materials and Methods: Two non-amplified near-infrared femtosecond lasers were used to irradiate porcine retinal specimens in vitro. The lasers were used for tissue removal as well as multiphoton laser scanning microscopy. Results: Ablation of the nerve fiber layer was performed at pulse energies of 1.0 nJ to 3.9 nJ. Control laser scanning images were acquired within seconds after irradiation. Specimens were additionally investigated with electron microscopy. Conclusions: Non-amplified femtosecond lasers may allow precise surgery controlled by fast high-resolution imaging of the target.

PURPOSE: To evaluate whether corneal flaps can be generated by the 80 MHz near-infrared, intense nanojoule femtosecond laser based on multiphoton absorption. METHODS: A solid-state Ti:Sapphire femtosecond laser system was integrated in an inverted JenLab Femt-O-Cut laser scanning microscope. A diffraction-limited 40X objective was used to induce multiphoton ionization and plasma production, New Zealand albino rabbits and porcine eyes were used. Surgical outcomes were determined using frame and line scans with nanojoule pulses at a wavelength of 800 nm. RESULTS: Surgical performance was assessed by optical imaging, histology, and electron microscopy. No significant corneal turbidity was observed. Optical imaging and histological examinations detected virtually no perturbation in the surrounding tissue. Corneal flaps and stromal lenticules were generated. Wound repair of the unlifted flaps was observed up to 90 days postoperatively. CONCLUSIONS: Surgical results and follow-up studies confirm that this femtosecond laser at nanojoule pulse energy is able to generate corneal flaps precisely, without causing visible collateral damage to the surrounding tissue or overlying epithelium.

Purpose: To investigate intravenous femtosecond laser surgery in models of branch retinal vein occlusion. Materials and Methods: Non-amplified near infrared femtosecond laser was used to ablate polyamide sutures and human hairs inserted into the vascular lumina of porcine retinal veins in vitro. Specimens were subjected to multiphoton laser scanning microscopy and electron microscopy. Results: Regular laser cuts within sutures and hairs were detected with laser microscopy and electron microscopy. Neither laser microscopy nor histology revealed collateral damage of the vascular wall. Conclusions: Non-amplified femtosecond lasers may allow precise atraumatic non-contact intravenous retinal surgery controlled by high-resolution imaging of the target.

We report on the influence of femtosecond laser pulses at different wavelengths (1035, 517, and 345 nm) on chinese hamster ovary cells exposed to intense radiation. The aim of the study was to determine the mean power thresholds from which the cells were influenced by the laser radiation up to irreversible cell damage. An influence of the wavelength has been shown. The damage threshold is lower in the ultraviolet (UV) than in the infrared (IR) by a factor of 5-6. At the green wavelength the threshold value was higher due to the low absorption (i.e., a relatively high transmission). The range between the lowest power where a loss of viability could be observed (25 mu W) and the mean power for direct cell damage (45 mu W) is 20 mu W in the UV. The corresponding range in the IR is between 75 and 240 mu W and thus about 165 mu W broad: In the case of UV irradiation, the cells seem to be unaffected close to the ablation or damage threshold at low energy pulses.

Non-invasive intratissue ablation was performed in the cornea of living rabbits by using 80 MHz near-infrared intense nanojoule femtosecond laser pulses. The intratissue surgical effect was induced by multiphoton absorption at a wavelength of 800 nm and was ascertained by histological examination. Highly precise intratissue ablation was obtained with no detrimental effects to the overlying or underlying layers. Activated keratocytes in the laser-treated corneas were detected with two-photon imaging postoperatively. Intratissue femtosecond laser ablation thus has potential as a effective technique in refractive surgery for the treatment of visual disorders.

Near-infrared multiphoton microscopes and in vivo femtosecond laser tomographs are novel powerful diagnostic tools for intra-tissue drug screening and high-resolution structural imaging applicable to many areas of biomedical research. Deep tissue cells and extracellular matrix (ECM) compartments can be visualized in situ with submicron resolution without the need for tissue processing. In particular, the reduced fluorescent coenzyme NAD(P)H, flavoproteins, keratin, melanin, and elastin are detected by two-photon excited autofluorescence, whereas myosin, tubulin and the ECM protein collagen can be imaged additionally by second harmonic generation (SHG). Therefore, these innovative multiphoton technologies have been used to probe architecture and state of a variety of native tissues, as well as of tissue-engineered constructs, giving insights on the interaction between scaffolds and seeded cells in vitro prior implantation. Moreover, non-invasive 4-D multiphoton tomographs are employed in clinical studies to examine the diffusion behavior, the intra-tissue accumulation of topically applied cosmetic and pharmaceutical components, and their interaction with skin cells.Near-infrared multiphoton microscopes and in vivo femtosecond laser tomographs are novel powerful diagnostic tools for intra-tissue drug screening and high-resolution structural imaging applicable to many areas of biomedical research. Deep tissue cells and extracellular matrix (ECM) compartments can be visualized in situ with submicron resolution without the need for tissue processing. In particular, the reduced fluorescent coenzyme NAD(P)H, flavoproteins, keratin, melanin, and elastin are detected by two-photon excited autofluorescence, whereas myosin, tubulin and the ECM protein collagen can be imaged additionally by second harmonic generation (SHG). Therefore, these innovative multiphoton technologies have been used to probe architecture and state of a variety of native tissues, as well as of tissue-engineered constructs, giving insights on the interaction between scaffolds and seeded cells in vitro prior implantation. Moreover, non-invasive 4-D multiphoton tomographs are employed in clinical studies to examine the diffusion behavior, the intra-tissue accumulation of topically applied cosmetic and pharmaceutical components, and their interaction with skin cells.