Biomedical subjects

Herbert Schneckenburger

Publications and source records attributed to Herbert Schneckenburger.

7 recordsLinked to original sources

PubMed2006

Microfluorometry of cell membrane dynamics.

Membranes of living cells are characterized by a combination of conventional and total internal reflection fluorescence microscopy (TIRFM) using the membrane marker laurdan. In the first case, all cellular membranes are assessed simultaneously, whereas in the second case, the plasma membrane is excited selectively by the evanescent electromagnetic field of a laser beam. A spectral shift depending on the phase of membrane lipids is used to characterize membrane stiffness, which decreases with temperature and increases with the amount of cholesterol. Spectral properties are evaluated and displayed as microscopic images.

2-Naphthylamine↗

PubMed2005

Total internal reflection fluorescence microscopy: technical innovations and novel applications.

Recent years have seen the introduction of novel techniques and applications of total internal reflection fluorescence microscopy (TIRFM). Key technical achievements include miniaturization, enhanced depth resolution, reduction of detection volumes and the combination of TIRFM with other microscopic techniques. Novel applications have concentrated on single-molecule detection (e.g. of cellular receptors), imaging of exocytosis or endocytosis, measurements of adhesion foci of microtubules, and studies of the localization, activity and structural arrangement of specific ion channels. In addition to conventional fluorescent dyes, genetically engineered fluorescent proteins are increasingly being used to measure molecular conformations or intermolecular distances by fluorescence resonance energy transfer.

Endocytosis↗

PubMed2004

Laser-assisted fluorescence microscopy for measuring cell membrane dynamics.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.

Animals↗

PubMed2004

Autofluorescence lifetime imaging of cultivated cells using a UV picosecond laser diode.

Lifetime images of autofluorescence of cultivated endothelial cells were recorded using a novel picosecond laser diode in the near ultraviolet range (375 nm). In contrast to existing picosecond light sources this wavelength permits efficient excitation of the free and protein bound coenzyme NADH with fluorescence lifetimes of 0.4-0.5 ns and 2.0-2.5 ns, respectively. The effective fluorescence lifetime tau(eff) (depending on both lifetimes) was homogenously distributed over the cells with some shortening in the perinuclear region, possibly close to mitochondria. A slight decrease of tau(eff) was observed after inhibition of the mitochondrial respiratory chain, whereas a slight increase was observed after inhibition of the glycolytic pathway, thus indicating variations of the ratio of free and protein bound NADH. Although present applications are still limited by their low pulse energy (< or = 5 pJ), uv picosecond laser diodes have a large potential in high resolution fluorescence microscopy and fluorescence lifetime endoscopy.

Adenosine Triphosphate↗

PubMed2004

Fluorescence lifetime imaging (FLIM) of rhodamine 123 in living cells.

A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolving techniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits optical excitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitation light is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7 endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine 123(R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria, whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly, fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of the R123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium should not be above 25 microM.

Animals↗

PubMed2002

Laser-assisted optoporation of single cells.

The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm(2) applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.

Animals↗

PubMedDate not supplied

Total internal reflectance fluorescence reader for selective investigations of cell membranes.

A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate (cell substrate), such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Main prerequisites are an appropriate thickness and a high transmission of the glass bottom, which is attached to the 96-well cell culture plate by a noncytotoxic adhesive. Glass rods of rectangular cross sections are optically coupled to this bottom for TIR illumination. Fluorescence arising from the plasma membrane of living cells is detected simultaneously from all samples using an integrating charge-coupled device (CCD) camera. The TIR fluorescence reader is validated using cultivated cells incubated with different fluorescent markers, as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard to potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells is determined.

Animals↗