Efficient cryopreservation of human pluripotent stem cells by surface-based vitrification.

Efficient cryopreservation of human stem cells is crucial for guaranteeing a permanent supply of high-quality cell material for drug discovery or regenerative medicine. Conventionally used protocols usually employing slow freezing rates, however, result in low recovery rates for human pluripotent stem cells due to their complex colony structure. In this chapter, a surface-based vitrification protocol for pluripotent stem cells is presented based on a procedure for human embryonic stem cells developed by Beier et al.

Laser scanning microscopy in cryobiology.

Laser scanning microscopy is emerging as a powerful imaging tool in cryobiology. The basic microscopy system can be combined with various imaging modalities including Raman spectroscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging, or multiphoton imaging. Multiphoton imaging can be used to study intracellular ice formation at the subcellular level.

Optical knock out of stem cells with extremely ultrashort femtosecond laser pulses.

Novel ultracompact multiphoton sub-20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two-photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two-photon imaging.

Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens.

We report on a nonlinear optical endoscope that adopts a hollow core photonic crystal fiber for single-mode illumination delivery and a multimode one for signal collection. Femtosecond laser pulses up to 100 mW can be delivered at a centered wavelength of 800 nm. The two-photon fluorescence response of our system is shown to have axial and lateral resolutions of 5.

Targeted transfection of stem cells with sub-20 femtosecond laser pulses.

Multiphoton microscopes have become important tools for non-contact sub-wavelength three-dimensional nanoprocessing of living biological specimens based on multiphoton ionization and plasma formation. Ultrashort laser pulses are required, however, dispersive effects limit the shortest pulse duration achievable at the focal plane. We report on a compact nonlinear laser scanning microscope with sub-20 femtosecond 75 MHz near infrared laser pulses for nanosurgery of human stem cells and two-photon high-resolution imaging.

Biosensor-based on-site explosives detection using aptamers as recognition elements.

Reliable observation, detection and characterisation of polluted soil are of major concern in regions with military activities in order to prepare efficient decontamination. Flexible on-site analysis may be facilitated by biosensor devices. With use of fibre-optic evanescent field techniques, it has been shown that immunoaffinity reactions can be used to determine explosives sensitively.

Mechanical properties of single cells by high-frequency time-resolved acoustic microscopy.

In this paper, we describe a new, high-frequency, time-resolved scanning acoustic microscope developed for studying dynamical processes in biological cells. The new acoustic microscope operates in a time-resolved mode. The center frequency is 0.

Imaging of focal contacts of chicken heart muscle cells by high-frequency acoustic microscopy.

A study of the adhesion of embryonic chicken heart muscle cells was conducted with a newly developed time-resolved acoustic microscope, which operates in the GHz-frequency range. The interpretation of the acoustical images of the heart muscle cells was done in combination with the fluorescence optical microscopy. A comparison between the acoustical images of chicken heart muscle cells and optical images of the same cells after staining showed that the actin fibers end inside the dark streaks in the acoustical images and thus represent the focal contacts (FCs).

Microfluidic patterning of alginate hydrogels.

In this article the authors present techniques which allow the microfluidic design of alginate microgels with layer composition on a chip. The hydrogel is created by combining two laminar flows of the gel precursor solutions-a calcium solution and an alginate solution-in a microchannel. The alginate solution is loaded with particles and by employing a certain fluid handling protocol involving several alginate solutions with different types of particles, a gel bar composed of many layers, each layer filled with a certain particle type, is formed.

Multiphoton microscopy for the investigation of dermal penetration of nanoparticle-borne drugs.

Multiphoton microscopy (MPM) of a dually fluorescence-labeled model system in excised human skin is employed for high-resolution three-dimensional (3D) visualization in order to study the release, accumulation, and penetration properties of drugs released from nanoscale carrier particles in dermal administration. Polymer particles were covalently labeled with fluorescein, whereas Texas Red as a drug-model was dissolved in the particles to be released to the formulation matrix. Single nanoparticles on skin could easily be localized and imaged with diffraction-limited resolution.

Optical nanoinjection of macromolecules into vital cells.

A tuneable near infrared 90MHz mode locked femtosecond laser was applied for targeted multiphoton optoporation of vital cells. Here, we demonstrate the laser assisted transfer of large amounts of exogenous materials and even macromolecules into living cells via a transient opening of the membrane and quantify the influx. The use of near infrared lasers also allow the optoporation of cells deep in three dimensional biological structures without photodestructive collateral effects.