Biomedical subjects

K O van der Werf

Publications and source records attributed to K O van der Werf.

10 recordsLinked to original sources

PubMed2006

Dependence of silicon position-detector bandwidth on wavelength, power, and bias.

We have developed a two-LED wobbler system to generate the spatial displacement of total light intensity on a detector surface, facilitating the acquisition of frequency responses up to 600 kHz with high accuracy. We have used this setup to characterize the low-pass filtering behavior of silicon-based position detectors for wavelengths above 850 nm by acquiring the frequency responses of several quadrant detectors and position-sensitive detectors as functions of wavelength, applied bias voltage, and total light power. We observed an increase in bandwidth for an increase in applied bias voltage and incident-light intensity. The combined effect of these parameters is strongly dependent on the detector used and has significant implications for the use of these detectors in scanning probe and optical tweezers applications.

Journal Article↗

PubMed2005

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology.

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.

Animals↗

PubMed1999

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.

Evaluation Studies as Topic↗

PubMed1998

Photon-counting device compatible with conventional flow cytometric data acquisition electronics.

We present an electronic scheme that enables us to use a photon-counting device (photomultiplier or avalanche photodetector) for measuring extremely weak signals in a flow cytometer. It can be used as a sole detector, or in combination with other (conventional) detectors using the data acquisition hardware of a conventional flow cytometer. The essential principle is that photon-counting pulses are converted to an analogue signal that is continuously proportional to the number of detected photons during the last integration time. The integration time should be approximately equal to the time an object is illuminated in the flow chamber. In this way, the photon burst due to real events is measured correctly and discriminated from the background pulses (fluorescence and Raman). The use of this scheme for the measurement of single DNA molecules is illustrated.

DNA↗

PubMed1998

Simultaneous height and adhesion imaging of antibody-antigen interactions by atomic force microscopy.

Specific molecular recognition events, detected by atomic force microscopy (AFM), so far lack the detailed topographical information that is usually observed in AFM. We have modified our AFM such that, in combination with a recently developed method to measure antibody-antigen recognition on the single molecular level (Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H. Schindler, Proc. Natl. Acad. Sci. USA 93:3477-3481 (1996)), it allows imaging of a submonolayer of intercellular adhesion molecule-1 (ICAM-1) in adhesion mode. We demonstrate that for the first time the resolution of the topographical image in adhesion mode is only limited by tip convolution and thus comparable to tapping mode images. This is demonstrated by imaging of individual ICAM-1 antigens in both the tapping mode and the adhesion mode. The contrast in the adhesion image that was measured simultaneously with the topography is caused by recognition between individual antibody-antigen pairs. By comparing the high-resolution height image with the adhesion image, it is possible to show that specific molecular recognition is highly correlated with topography. The stability of the improved microscope enabled imaging with forces as low as 100 pN and ultrafast scan speed of 22 force curves per second. The analysis of force curves showed that reproducible unbinding events on subsequent scan lines could be measured.

Aluminum Silicates↗

PubMed1998

Direct visualization of dynamic protein-DNA interactions with a dedicated atomic force microscope.

Photolyase DNA interactions and the annealing of restriction fragment ends are directly visualized with the atomic force microscope (AFM). To be able to interact with proteins, DNA must be loosely bound to the surface. When MgCl2 is used to immobilize DNA to mica, DNA is attached to the surface at distinct sites. The pieces of DNA in between are free to move over the surface and are available for protein interaction. After implementation of a number of instrumental improvements, the molecules can be visualized routinely, under physiological conditions and with molecular resolution. Images are acquired reproducibly without visible damage for at least 30 min, at a scan rate of 2 x 2 microm2/min and a root mean square noise of less than 0.2 nm. Nonspecific photolyase DNA complexes were visualized, showing association, dissociation, and movement of photolyase over the DNA. The latter result suggests a sliding mechanism by which photolyase can scan DNA for damaged sites. The experiments illustrate the potential that AFM presents for modern molecular biology.

Binding Sites↗

PubMed1998

Tuning fork shear-force feedback.

Investigations have been performed on the dynamics of a distance regulation system based on an oscillating probe at resonance. This was examined at a tuning fork shear-force feedback system, which is used as a distance control mechanism in near-field scanning optical microscopy. In this form of microscopy, a tapered optical fiber is attached to the tuning fork and scanned over the sample surface to be imaged. Experiments were performed measuring both amplitude and phase of the oscillation of the tuning fork as a function of driving frequency and tip-sample distance. These experiments reveal that the resonance frequency of the tuning fork changes upon approaching the sample. Both the amplitude and the phase of the tuning fork can be used as distance control parameter in the feedback system. Using the amplitude a second-order behavior is observed, while with phase only a first-order behavior is observed. Numerical calculations confirm these observations. This first-order behavior results in an improved stability of the feedback system. As an example, a sample consisting of DNA strands on mica was imaged which showed the height of the DNA as 1.4 +/- 0.2 nm.

Aluminum Silicates↗

PubMed1994

Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy.

Application of atomic force microscopy (AFM) to biological objects and processes under physiological conditions has been hampered so far by the deformation and destruction of the soft biological materials invoked. Here we describe a new mode of operation in which the standard V-shaped silicon nitride cantilever is oscillated under liquid and damped by the interaction between AFM tip and sample surface. Because of the viscoelastic behavior of the cellular surface, cells effectively "harden" under such a tapping motion at high frequencies and become less susceptible to deformation. Images obtained in this way primarily reveal the surface structure of the cell. It is now possible to study physiological processes, such as cell growth, with a minimal level of perturbation and high spatial resolution (approximately 20 nm).

Animals↗

PubMed1993

A simple optical fiber device for quantitative fluorescence microscopy of single living cells.

A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is 'opened' and 'closed' can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-loaded K562 target cells bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp.

Calcium↗

PubMedDate not supplied

Analysis of immunolabeled cells by atomic force microscopy, optical microscopy, and flow cytometry.

In this study we investigated the applicability of the (silver-enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied on these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a direct correlation was made between the force and the fluorescent images. Additionally, we performed flow cytometric analysis. From the results we conclude that immunogold labeling using small labels (1 nm) in combination with silver enhancement (30 min) proves to be a reliable method for high-resolution cell surface antigen detection in atomic force microscopy.

Animals↗