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H C Gerritsen

Publications and source records attributed to H C Gerritsen.

11 recordsLinked to original sources
PubMed2002

Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution.

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two- to eight-channel, time-gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time-gates are significantly more sensitive than the two time-gate system. Only minor sensitivity differences were found between systems with four or more time-gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP-PMTs and 300-400 ps for other detectors. The maximum count rate of fast MCP-PMTs, however, is 10-100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP-PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.

Animals↗
PubMed2001

Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.

Fluorescence↗
PubMed1999

Imaging of optically thick specimen using two-photon excitation microscopy.

The in-depth imaging properties of two-photon excitation microscopy were investigated and compared with those of confocal microscopy. Confocal imaging enabled the recording of images from dental biofilm down to a depth of 40 microm, while two-photon excitation images could be recorded at depths greater than 100 microm. Two-photon excitation point spread functions (PSFs) were recorded at depths ranging from 0 to 90 microm depth using 220-nm diameter fluorescent beads immersed in water. PSFs were measured using both a high numerical aperture oil immersion objective and a water immersion objective. The experiments carried out using the oil immersion objective showed a rapid degradation of both the axial and lateral resolution due to spherical aberrations. In addition, the detected fluorescence intensity rapidly decreased as a function of depth. The experiments carried out using the water immersion objective showed no significant degradation of both the axial and lateral resolution and the fluorescence intensity.

Bacteria↗
PubMed1999

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.

Biofilms↗
PubMed1996

High-resolution confocal microscopy using synchrotron radiation.

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper.

Animals↗
PubMed1995

Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy.

The pH-sensitive probe carboxy SNAFL-1 can be used for imaging using ratiometric and fluorescence lifetime techniques. The former method suffers from the drawback that quantitative pH imaging in cells requires a time-consuming and cumbersome calibration procedure. In contrast, straightforward calibrations in buffer suffice for fluorescence lifetime imaging. This is illustrated here by a comparative study of the two techniques under different controlled conditions. The effect of probe concentration, protein concentration, and hydrophobicity, the contents of damaged cells and living cells on the emission ratio, and the fluorescence lifetime of carboxy SNAFL-1 were studied. The results clearly demonstrate that the fluorescence lifetime imaging technique is more convenient than the ratiometric method for pH determination.

Animals↗
PubMed1994

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres.

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S1 during the transition.

Animals↗
PubMed1994

[Application of confocal laser microscopy in studying the effect of epidermal growth factor on the seminiferous epithelium].

Effects of epidermal growth factor (EGF) on pH transients in aggregates of Sertoli cells and germinal cells have been investigated with confocal microscopy using a fluorescent pH sensitive indicator. In some Sertoli cells EGF caused a rapid rise in the pH whereas other Sertoli cells did not respond to EGF. Some Sertoli cells showed a delayed response which coincidated with a similar pH change in neighbouring germinal cells. Since isolated germinal cells never showed a pH response after exposure to EGF, we have concluded that some Sertoli cells may communicate with germinal cells via gap junctions.

Animals↗
PubMed1992

The orientation of transition moments of dye molecules used in fluorescence studies of muscle systems.

Fluorescence and phosphorescence depolarization techniques can provide information on orientational order and rotational motion of crossbridges in muscle fibres. However the depolarization experiment monitors the orientation and motion of the crossbridges indirectly. The changes in depolarization arise from a change in the orientation of the transition dipoles of the dye attached to the crossbridge. In order to extract the physiologically relevant orientations from the data it is therefore necessary to characterize the orientation of the dye molecule relative to the crossbridge and the orientation of the transition moments in the frame of the dyes. The dyes 1,5-I-AEDANS and eosin-5-maleimide are commonly used for labelling the crossbridge in muscle fibres. The orientations of the absorption and fluorescence emission dipoles of these two dyes in the molecular frame were determined. Angle resolved fluorescence depolarization experiments on the dyes, macroscopically aligned in a stretched polymer matrix of poly vinyl alcohol, were carried out. The data were analyzed in terms of an orientational distribution of the dye molecules in the film and the orientations of the absorption and emission dipoles in the frame of the dye molecule. Experimental data, obtained from a given sample at different excitation wavelengths, were analyzed simultaneously in a global target approach. This leads to a reduction in the number of independent parameters optimized by the non-linear least squares procedure.

Animals↗
PubMedDate not supplied

Fast fluorescence lifetime imaging of calcium in living cells.

A fast fluorescence lifetime imaging (FLIM) system is developed that can acquire images at a rate of hundreds of frames per second. The FLIM system is based on a wide-field microscope equipped with a time-gated intensified CCD detector and a pulsed laser. The time-gated detector acquires the signals from two time gates simultaneously and is therefore insensitive to movements of the specimen and photo-bleaching. The system is well suited for quantitative biological FLIM experiments and its performance is evaluated in calcium imaging experiments on beating neonatal rat myocytes. Several calcium sensitive dyes are characterized and tested for their suitability for fast FLIM experiments: Oregon Green Bapta-1 (OGB1), Oregon Green Bapta-2 (OGB2), and Oregon Green Bapta-5N (OGB5N). Overall the sensitivity range of these dyes is shifted to low calcium concentrations when used as lifetime dyes. OGB1 and OGB2 behave very similarly and can be used for FLIM-based calcium imaging in the range 1 to approximately 500 nM and OGB5N can be used up to 3 microM. The fast FLIM experiments on the myocytes could be carried out at a 100-Hz frame rate. During the beating of the myocytes a lifetime change of about 20% is observed. From the lifetime images a rest calcium level of about 65 nM is found.

Algorithms↗