Alterations in Human Liver Metabolome during Prolonged Cryostorage.

Tissue metabolomics requires high sample quality that crucially depends on the biobanking storage protocol. Hence, we systematically analyzed the influence of realistic storage scenarios on the liver metabolome with different storage temperatures and repeated transfer of samples between storage and retrieval environments, simulating the repeated temperature changes affecting unrelated samples stored in the same container as the sample that is to be retrieved. By cycling between storage (-80 °C freezer, liquid nitrogen, cold nitrogen gas) and retrieval (room temperature, -80 °C), assuming three cycles per day and sample, we simulated biobank storage between 3 months and 10 years.

Ultraviolet femtosecond laser creation of corneal flap.

Purpose: To study parameters for ocular femtosecond laser surgery in terms of process efficiency and safety aspects using ultraviolet (UV) femtosecond laser pulses.

Methods: Studies on corneal surgery and flap processing on enucleated porcine eyes were performed using a newly developed ytterbium-doped gain media laser source. Ultraviolet femtosecond laser pulses centered at a wavelength of 345 nm and working at a repetition rate of 100 kHz were generated by the third harmonics of the 1035-nm fundamental wavelength.

Two-photon autofluorescence and second-harmonic imaging of adult stem cells.

Human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells) are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation (SHG) are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H] and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids.

Clinical two-photon microendoscopy.

Two-photon medical imaging has found its way into dermatology as an excellent method for noninvasive skin cancer detection without need of contrast agents as well as for in situ drug screening of topically-applied cosmetical and pharmaceutical components. There is an increasing demand to apply the multiphoton technology also for deep-tissue skin imaging as well as for intracorporal imaging. We report on the first clinical use of multiphoton endoscopes, in particular of a miniaturized rigid two-photon GRIN lens endoscope.

Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging.

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules.

Multiphoton fluorescence lifetime imaging of human hair.

In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.

In vivo drug screening in human skin using femtosecond laser multiphoton tomography.

The novel femtosecond laser multiphoton imaging system DermaInspect forin vivotomography of human skin was used to study the diffusion and intradermal accumulation of topically applied cosmetic and pharmaceutical components. Near-infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores and fluorescent components of a variety of ointments via a two-photon excitation process. In addition, collagen was imaged by second harmonic generation.

Multiphoton autofluorescence imaging of intratissue elastic fibers.

Multiphoton induced blue/green autofluorescence by near infrared femtosecond laser pulses has been used to selectively image intratissue elastic fibers in native and tissue engineered (TE) viable heart valves without any invasive tissue removal, embedding, fixation, and staining. Elastic fibers could be clearly distinguished from collagenous structures which emit ultraviolet/violet radiation when excited with intense ultrashort pulses due to second harmonic generation. Deep-tissue three-dimensional imaging of elastic fibers with submicron spatial resolution was performed by optical sectioning of heart valves using a multiphoton laser scanning microscope in connection with a tunable 80 MHz femtosecond laser source.