Ernst-Ludwig Florin

Sensing specific molecular interactions with the atomic force microscope

One of the unique features of the atomic force microscope (AFM) is its capacity to measure interactions between tip and sample with high sensitivity and unparalleled spatial resolution. Since the development of methods for the functionalization of the tips, the versatility of the AFM has been expanded to experiments where specific molecular interactions are measured. For illustration, we present measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. Here, we compare different techniques that allow for the in situ measurement of the absolute value of the spring constant of cantilevers.

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

We use single-particle tracking to study the elastic properties of single microtubules grafted to a substrate. Thermal fluctuations of the free microtubule’s end are recorded, in order to measure position distribution functions from which we calculate the persistence length of microtubules with contour lengths between 2.6 and 48 μm. We find the persistence length to vary by more than a factor of 20 over the total range of contour lengths. Our results support the hypothesis that shearing between protofilaments contributes significantly to the mechanics of microtubules. PACS numbers: 87.15.Ya, 87.15.La, 87.16.Ka, 36.20.Ey ∗ Corresponding author. FP and GL have equally contributed to this work.

Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging

We show that the position of a fluorescent nanoparticle can be measured in three dimensions with subnanometer precision and 100-ms temporal resolution by use of standard epifluorescence video imaging in off-focus mode. The particle can be tracked without feedback in a volume of at least 40 microm x 60 microm x 3 microm. With the technique presented, the structure-mobility relationship of 216-nm latex particle in a porous polymer network was studied in three dimensions.

Collective motion and density fluctuations in bacterial colonies

Flocking birds, fish schools, and insect swarms are familiar examples of collective motion that plays a role in a range of problems, such as spreading of diseases. Models have provided a qualitative understanding of the collective motion, but progress has been hindered by the lack of detailed experimental data. Here we report simultaneous measurements of the positions, velocities, and orientations as a function of time for up to a thousand wild-type Bacillus subtilis bacteria in a colony. The bacteria spontaneously form closely packed dynamic clusters within which they move cooperatively. The number of bacteria in a cluster exhibits a power-law distribution truncated by an exponential tail. The probability of finding clusters with large numbers of bacteria grows markedly as the bacterial density increases. The number of bacteria per unit area exhibits fluctuations far larger than those for populations in thermal equilibrium. Such “giant number fluctuations” have been found in models and in experiments on inert systems but not observed previously in a biological system. Our results demonstrate that bacteria are an excellent system to study the general phenomenon of collective motion.

Trapping and tracking a local probe with a photonic force microscope

An improved type of scanning probe microscope system able to measure soft interactions between an optically trapped probe and local environment is presented. Such a system that traps and tracks thermally fluctuating probes to measure local interactions is called a photonic force microscope (PFM). The instrument can be used to study two-dimensional and three-dimensional surface forces, molecular binding forces, entropic and viscoelastic forces of single molecules, and small variations in particle flow, local diffusion, and viscosities. We introduce and characterize a PFM, and demonstrate its outstanding stability and very low noise. The probe’s position can be measured within a precision of 0.2–0.5 nm in three dimensions at a 1 MHz sampling rate. The trapping system facilitates stable trapping of latex spheres with diameter D=λ0/2 at laser powers as low as 0.6 mW in the focal plane. The ratio between the trapping stiffness and laser power was able to be optimized for various trapping conditions. The measur...

Painted supported lipid membranes

We report herein measurements on a novel type of supported lipid films, which we call painted supported membranes (PSM). These membranes are formed in a self-assembly process on alkylated gold films from an organic solution. The formation process was investigated with surface plasmon resonance microscopy. The optical and electrical properties of the films were determined for various types of lipids and as a function of temperature by means of cyclic voltammetry and potential relaxation after charge injection. We could show that these films exhibit an extraordinarily high specific resistivity which, depending on the lipid, may be as high as 10(9) ohm/cm(2). We could also show that due to this low conductivity, an electrical polarization across the PSM relaxes with characteristic time constants of up to 20 min. The electrical properties together with their high mechanical stability and accessibility to surface sensitive techniques make these films well suitable model membranes for optical and electrical investigations. Examples for such applications are given in the subsequent article by Seifert et al.

Atomic force microscope with magnetic force modulation

We have constructed a scanned stylus atomic force microscope (AFM) with direct force modulation and integrated microfluorescence optics. The instrument was designed to image the surface of massive samples under various ambient conditions. In force modulation microscopy the imaging force is modulated during the scanning process via an external magnetic field that acts directly on the magnetic AFM tip. Polymeric Langmuir–Blodgett films on silicon oxide were imaged to evaluate the application range of the instrument. We demonstrate that direct force modulation microscopy permits the quantitative recording of the local complex compliance both as a function of the location and as a function of the frequency. In a novel imaging mode referred to as sample resonance mode, the contrast of the image can be selectively enhanced based on local elasticity differences.

Adhesive forces between ligand and receptor measured by AFM

Abstract Since its invention, the atomic force microscope (AFM) has been used to image a wide range of samples, including soft biological materials. Although various imaging modes have been developed in recent years, the images obtained by these techniques are primarily derived from the mechanical interactions between AFM tip and sample. An imaging mode based on the specific recognition of defined functional groups on the sample by receptor molecules attached to the AFM tip may prove to be beneficial, especially in complex specimens such as the cell membrane. A prerequisite towards this goal is the development of AFM tips functionalized with ligand-specific receptors. Here we report the fabrication of tips functionalized with either avidin or antibodies. These functionalized tips were characterized by force scan measurements on ligands cross-linked to agarose beads. The specificity of the observed adhesion between tip and sample was confirmed by specific blocking of the ligands or the receptors.

Microtubule architecture: inspiration for novel carbon nanotube-based biomimetic materials

Microtubules are self-assembling biological nanotubes that are essential for cell motility, cell division and intracellular trafficking. Microtubules have outstanding mechanical properties, combining high resilience and stiffness. Such a combination allows microtubules to accomplish multiple cellular functions and makes them interesting for material sciences. We review recent experiments that elucidate the relationship between molecular architecture and mechanics in microtubules and examine analogies and differences between microtubules and carbon nanotubes, which are their closest equivalent in nanotechnology. We suggest that a long-term goal in bionanotechnology should be mimicking the properties of microtubules and microtubule bundles to produce new functional nanomaterials.

Unintended filtering in a typical photodiode detection system for optical tweezers

We characterize the frequency-dependent response of a photo detection system based on a Si-PIN photodiode and a laser with wavelength 1064 nm, a system commonly used with optical tweezers. We chopped the laser beam with chopper frequencies from 200 Hz to 14 kHz, and found an exponentially delayed response of the detection system with a characteristic delay time of ∼20 μs. The physical mechanism causing this time delay is silicon’s transparency to 1064 nm light: Photons are absorbed and create charge carriers not only in the diode’s depletion layer, where they are detected within nano-seconds, but predominantly in the n-layer, where they remain undetected till transported out by thermal diffusion. The diode’s response is dominated by this delay which can be characterized as a first-order low-pass filter with a 3dB-frequency of 8–9 kHz, depending on laser intensity. Measurements exploiting frequencies near or above this 3dB-frequency must be corrected for this unintended filter effect. We describe how to do...