Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers.

Single chromatin fibers were assembled directly in the flow cell of an optical tweezers setup. A single lambda phage DNA molecule, suspended between two polystyrene beads, was exposed to a Xenopus laevis egg extract, leading to chromatin assembly with concomitant apparent shortening of the DNA molecule. Assembly was force-dependent and could not take place at forces exceeding 10 pN. The assembled single chromatin fiber was subjected to stretching by controlled movement of one of the beads with the force generated in the molecule continuously monitored with the second bead trapped in the optical trap. The force displayed discrete, sudden drops upon fiber stretching, reflecting discrete opening events in fiber structure. These opening events were quantized at increments in fiber length of approximately 65 nm and are attributed to unwrapping of the DNA from around individual histone octamers. Repeated stretching and relaxing of the fiber in the absence of egg extract showed that the loss of histone octamers was irreversible. The forces measured for individual nucleosome disruptions are in the range of 20-40 pN, comparable to forces reported for RNA- and DNA-polymerases.

Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies.

BACKGROUND: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.

Biomolecular interactions measured by atomic force microscopy.

Atomic force microscopy (AFM) is nowadays frequently applied to determine interaction forces between biological molecules. Starting with the detection of the first discrete unbinding forces between ligands and receptors by AFM only several years ago, measurements have become more and more quantitative. At the same time, theories have been developed to describe and understand the dynamics of the unbinding process and experimental techniques have been refined to verify this theory. In addition, the detection of molecular recognition forces has been exploited to map and image the location of binding sites. In this review we discuss the important contributions that have led to the development of this field. In addition, we emphasize the potential of chemically well-defined surface modification techniques to further improve reproducible measurements by AFM. This increased reproducibility will pave the way for a better understanding of molecular interactions in cell biology.

Dynamic regulation of activated leukocyte cell adhesion molecule-mediated homotypic cell adhesion through the actin cytoskeleton.

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM-ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM-ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.

Optical tracking and detection of immunomagnetically selected and aligned cells.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

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.

Orientation of the chromophore dipoles in the TOTO-DNA system.

BACKGROUND: Flow cytometry has been applied successfully to the sizing of medium to large-sized DNA molecules, thanks to the excellent staining properties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (Petty et al.: Anal Chem 67:1755-1761, 1995). The hydrodynamic flow, used to focus the sample molecules in a small laser-illuminated volume, is also responsible for their alignment, thereby allowing the determination of the TOTO-dipole orientation with respect to the DNA axis (Agronskaia et al.: Appl Opt 38:714-719, 1999). METHODS: We present model calculations of the fluorescence yield of TOTO-stained DNA measured in a flow-cytometric setup with high numerical aperture. The models consider different orientations of the chromophore dipoles. RESULTS: Comparison of measurement and calculation suggests that the absorption dipoles of the TOTO molecule make a mean angle of 61 degrees with the helix axis of the DNA molecule. This mean angle can be the consequence of two binding modes. CONCLUSIONS: Our results indicate that any model with a significant contribution of perpendicularly-oriented chromophores fails to reproduce the experimental results.

High speed atomic force microscopy of biomolecules by image tracking.

An image-tracking procedure for atomic force microscopy is proposed and tested, which allows repeated imaging of the same area without suffering from lateral drift. The drift correction procedure is based on on-line cross-correlation of succeeding images. Using the image-tracking procedure allows zooming in on a small scan area over a long period and thus increases the frame rate inversely proportional to the scan area. Application of the procedure is demonstrated for diffusion of 5.4-kb DNA plasmids. With a scan area of 500 * 500 nm(2), a single plasmid can be imaged for more than 30 min at 4 s per frame, with a drift less than 10 nm. The high temporal resolution allows detailed analysis of the diffusion of DNA molecules. A diffusion coefficient of 30 nm(2)/s is found for most DNA molecules, though many molecules are temporally pinned to the mica surface, restricting diffusion.

3D single-particle tracking and optical trap measurements on adhesion proteins.

A three-dimensional single-particle tracking system was combined with an optical trap to investigate the behavior of transmembrane adhesion proteins. We exploited this setup to investigate which part of the cell adhesion protein LFA-1 forms a connection to the cytoskeleton after binding to its ligand ICAM-1. LFA-1 is an integrin consisting of an alpha and a beta chain. Thus far, only the cytoplasmic tail of the beta chain is known to form a connection to the cytoskeleton. We investigated cells that express a mutant form of LFA-1 that lacks the complete beta cytoplasmic tail and therefore is not thought to bind to the cytoskeleton. Interestingly, single-particle tracking measurements using beads coated with the ligand ICAM-1 indicate that this mutant form of LFA-1 does not move freely within the cell membrane, suggesting that LFA-1 is still connected to the cytoskeleton network. This finding is strongly supported by the observation that LFA-1 exhibits a more diffusive motion when the cytoskeleton network is disrupted and confirmed by the optical trap measurements used to force the proteins to move through the membrane. Collectively, our findings suggest that the interaction of LFA-1 with the cytoskeleton cannot solely be attributed to the cytoplasmic part of the beta chain.

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.

Visualising individual green fluorescent proteins with a near field optical microscope.

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximately 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutants was monitored in time, with 500-micros real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-off behaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements.

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy.

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.

Polarization effects in flow cytometric DNA sizing.

We have investigated the influence of the polarization direction of excitation light on DNA sizing results obtained by a flow cytometric technique. We found strong fluorescence anisotropy of the fluorescent signal from lambda DNA stained with the bis-intercalating dye TOTO-1. Small fragments of DNA are less sensitive to polarization than larger pieces. This effect is more pronounced at faster flow speeds. These observations show a noticeable orientation of the DNA molecules introduced by the shear forces in the flow system. The data are consistent with an angle between the transition moment of fluorescence of TOTO-1, and the long axis of DNA is approximately 62 degrees .

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.

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.

New technique for high resolution DNA sizing in epi-illumination.

We present a high-resolution DNA-sizing technique based on the principles of flow cytometry, using a high numerical aperture objective and epi-illumination. The new technique, designed for small fluorescing samples/particles (sub-micron diameter) suspended in a weakly fluorescent medium, makes use of an additional focus for high-precision particle localisation. This way, only those particles are considered that flow exactly through a well-defined volume. Results are presented for fluorescent beads, as well as for YOYO-stained plasmids containing 5,500 basepairs. The latter were measured with 6.2% resolution, setting a new limit to flow-based sizing of DNA.

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.

Experimental and model investigations of bleaching and saturation of fluorescence in flow cytometry.

We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein-isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate kb1 = 6 x 10(3) s(-1), triplet state population rate k12 = 2 x 10(5) s(-1), and depopulation rate k20 = 5 x 10(4) s(-1). The fluorescence kinetics of FITC- and PE-labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water.

Imaging of RNA in situ hybridization by atomic force microscopy.

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3.3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA--in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy.

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.