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  • Biophysics & Nanosciences - „Physics of Life“

    Scanning electron microscopy
    Scanning electron microscopy © Universität Bielefeld/Christoph Pelargus

Scanning probe microscopy

Atomic Force Microscopy (AFM)

Atomic Force Microscopy (AFM)

STRUCTURAL BIOLOGY

Contact: Dr. Volker Walhorn

AFM scan from a cell of the lung tissue
Cell of the lung tissue - © Universität Bielefeld/Daniel Wesner

Structural information of individual functional molecules and complexes can be investigated by in-situ atomic force microscopy (AFM) by immobilising the biomolecules of interest (nucleic acids, proteins, carbohydrates, …) via physical, chemical or biological interaction on a flat surface or directly embedded in the cellular membrane environment. The interesting structural information includes sub-nm-conformation, molecular symmetry, binding location and molecular temporal dynamics. The following typical examples are typical results of actual research projects where biological processes of higher complexity (transcription regulation,molecular motors, self-assembly of 2D-protein s-layers) are investigated.
We currently run several commercial AFM for experiments under ambient and liquid conditions, self-built combined TIRF/AFM and SNOM microscopes, as very recently a novel STM/AFM-microscope for ultra-high-vacuum applications.

 

 

Atomic force scan of ordered diyne-phosphocholine molecules
© Universität Bielefeld/Fabian Heising

Diyne-phosphocholine molecules arrange themselves after Langmuir-Blodgett transfer onto Highly Ordered Pyrolytic Graphite - a step towards simulating bilipid cell membranes.

Scan of a proteoglycan molecule from the sea cucumber Isostichopus badionotus with AFM
© Universität Bielefeld/Niklas Biere

A proteoglycan molecule from the sea cucumber Isostichopus badionotus has a brush-like structure. The delicate arms can bind water and this way change the mechanical properties of their tissue.

AFM picture from fibroblast cell from human tissue
© Universität Bielefeld/Orooba Al-Hammood

This fibroblast cell from human tissue is fixated and prepared for force spectroscopic measurements - to determine its elastic properties and ultimately their relation to genetic heart diseases.

Single Molecule AFM Force Spectroscopy

PHYSICS OF MOLECULAR RECOGNITION

Contact: Dr. Volker Walhorn

AFM head with equipment
Flowbox workbench - © Universität Bielefeld/Niklas Biere

The physical mechanisms of specific, non-covalent intermolecular binding is quantitatively investigated by single-molecule (dynamic) force spectroscopy with atomic force microscopy (AFM), optical tweezers (OT) and magnetic tweezers (MT). Here, specific molecular forces, elasticities, kinetic reaction rate constants (lifetimes) and the energy landscape of the molecular binding potential is measured by AFM at the single-molecule level in vitro on isolated but functional complexes. Nowadays, these specific interactions can be scrutinized in a quantitative manner at the sensitivity level on single-point mutations (nucleic acids, amino acids) allowing single-molecule affinity ranking in a broad affinity range of 0,1 mM-1 fM revealing distinct differences in the binding properties and mechanisms. Over the last years, special emphasis has been put on so-called molecular catch-bond systems that were observed when investigating the interplay of human sulfatates (Sulf1, Sulf2) with glycosaminoglycans (heparan sulfate, heparin, dermatan sulfate, ...) in cell signaling cascades. In addition, we recently explored the homophilic interaction mechanisms between desmosomal desmoglein-2 (DSG2) and mutations thereof that play an important role for the integrity of mechanically active cells like cardiomyocytes.

Schematic of how atomic force microscopy works
© Universität Bielefeld/Niklas Biere

Tip and sample surface are both functionalized. In close proximity, individual molecular bonds are formed. When pulled apart, ruptures can be seen as a jumps in the force curve. The corresponding binding forces and lifetimes can be measured with high precision, giving insight in biological relevant binding and sensing processes and even protein folding.

A merged phase contrast and fluorescence microscopy image of a cell
© Universität Bielefeld/Esther Borchers

Fluorescent labeled cell compartments help guide where to poke the cell - to measure the elasticity in regards to specific organelles that might be affected by genetic conditions.

Single Molecule Photonics

AFM-TIRF / SNOM

Contact: Dr. Volker Walhorn

 Self made TIRF combined AFM head
Self made TIRF combined AFM head - © Universität Bielefeld/C.Pelargus

Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Single molecule fluorescence imaging and spectroscopy yield valuable and quantitative information about optical properties, spatial distribution and temporal dynamics of single molecules.
We have developped 1) scanning near-field optical microscopy (SNOM) for single molecule imaging and spectroscopy and 2) a small-cantilever based AFM combined with total internal reflection fluorescence (TIRF) microscopy for ultrasensitive LIF detection of individual fluorophores and simultaneous AFM control and manipulation at the molecular level.

A SNOM scan of  tobaco viruses
© Universität Bielefeld/Niklas Biere

Individual fluorescent markers can be localized in the AFM topography with super resolution precision - here shown in tobacco mosaic viruses.

Simulation of an electric field at a gold tip 5nm above an air-glass interface
© Universität Bielefeld/Volker Walhorn

Simulation of an electric field at a gold tip 5nm above an air-glass interface. The configuration is illuminated from below at an angle of 50°, so that an evanescent field is formed above the interface
 

3D represntation of the principal function of evanescent illumination
© Universität Bielefeld/Niklas Biere

Evanescent illumination causes several hundred-times higher field intensities at the cantilever apex as usual - also enhancing the radiative emission of fluorophor

Nanomanipulation

Nanopores

Nanopores

SINGLE MOLECULE TRANSLOCATION

Contact: Prof. Dr. Dario Anselmetti

SEM grid for preparation
SEM grid for preparation - © Universität Bielefeld/Roland Hillmann

The translocation dynamics of individual macromolecules like DNA or DNA-protein complexes (EcoRI, RecA, peroxiredoxines) through solid-state nanopores (SiNx, graphene, MoS2, BN, ...) is quantitativly investigated by optical tweezers force control. Beyond looking into the physical mechanisms involved we explore the possibility of future sequencing and diagnostic applications thereof.

Pore cover from backside ,optical microscope view, while evaporating the solvent.
© Universität Bielefeld/Dennis Kreft

Pore cover from backside ,optical microscope view, while evaporating the solvent.

Freestanding MoS2-monolayer with a thickness of 0.67nm covering a 600nm opening
© Universität Bielefeld/Julian Cremer

Freestanding MoS2-monolayer with a thickness of 0.67nm covering a 600nm opening

Optical Tweezers

SINGLE MOLECULE BIOSENSOR & MANIPULATION

Contact: Prof. Dr. Dario Anselmetti

 Glass capillary
Glass capillary - © Universität Bielefeld/Tobias Jäckering

Micron-sized objects like beads, colloids or cells can be trapped, steered and manipulated by light and allow force experiments at the single molecule level with a force sensitivity level of 0.1 pN. We set up two high stability single-beam optical tweezers (OT) system on an inverted light microscope which allows analytical force spectroscopy, adhesion and elasticity experiments with single molecules or cells in an experimental force range of 0.1-900 pN.

Principle in the experiments with controlled DNA translocations through nanopores with optical tweezers
© Universität Bielefeld/Lukas Galla

Principle in the experiments with controlled DNA translocations through nanopores with optical tweezers.

Measuring the elasticity of a HEK-293 cell with two optically trapped polystyrene beads
© Universität Bielefeld/Sebastian Knust

Measuring the elasticity of a HEK-293 cell with two optically trapped polystyrene beads

Magnetic Tweezers

SINGLE MOLECULE MANIPULATION WITH MAGNETIC FIELDS

Contact: Prof. Dr. Dario Anselmetti

Sample of fluids in test tubes
Different buffer concentrations © Universität Bielefeld/Niklas Biere

Magnetic microbeads can be manipulated and steered by external magnetic fields and allow stretching and overwinding experiments with single (DNA) molecules at a sensitivity level downto 0,001 pN. With our magnetic tweezers (MT) apparatus (PicoTwist, Lyon - France) we investigate and quantify the binding of proteins, fluorescent dyes and chemotherapeutic agents to DNA.

Magnetic beads
© Universität Bielefeld/Marcel Skaletz

Magnetic beads are optically transparent. Their diffraction pattern is used to determine their vertical position and the length of the attached DNA stands

Microfluidics

Microfluidics

Micro- & Nanofluidics

MIGRATION & DYNAMICS OF BIOMOLECULES

Contact: Dr. Martina Viefhues

Dielectrophoretic separation of DNA on a microfluidic chip
PDMS chip with electrodes - © Universität Bielefeld/C.Pelargus

In this research project we investigate and develop novel micro- and nanofluidical chip device applications that can be used for separation, sorting and harvesting of macromolecules and colloids. We use structured micro- and nanofluidic environments and drive the molecular objects far from thermoddynamic equilibrium to explore non-linear migration mechanisms like absolute negative mobility, dielectrophoretic trapping, molecular ratcheting or chiral separation.
In addition, we design and develop lab-on-a-chip devices where individual cells like E. coli can be trapped and analyzed according their active metabolism by UV-laser-induced fluorescence.

Microscopy picture of a 20µm width PDMS microchanel crossing with a cell trap
© Universität Bielefeld/Armin Grundmann

Microscopy picture of a 20µm width PDMS microchanel crossing with a cell trap.

Microfluidic flow cell with high voltage electrodes conducted and fluorescence detection
© Universität Bielefeld/Christoph Pelargus

Microfluidic flow cell with high voltage electrodes conducted and
fluorescence detection.

Tribologie

Industrial Projects (BINAS)

Industrial Projects (BINAS)

NANOPARTICLES, TRIBOLAB, ...

Contact: Dr. Katja Tönsing

Mikroscopy picture of a typical steel friction test sample
Test disc with worn surface - © Universität Bielefeld/Alexander Beel

In this research and development project we investigate and develop new methods and products for use in industrial applications. Together with our industrial partners we analyze, develop and optimize nanotechnological products and processes ranging from e.g. nanoparticle based lubricating performance systems, nanomembrane-based biosensor surfaces as well as effective microchip processes for separating large genomic mixtures.

Test samples with marbles made of hardened steel for friction measurement
© Universität Bielefeld/Niklas Biere

Test samples with marbles made of hardened steel for friction measurement. Different oil mixtures can be testet.



Diverses

  Thema Ansprechpartener Infos
Bachelorarbeiten / Abschlussarbeiten fürs Lehramt ... nach Absprache Prof. Dario Anselmetti (D1-270) Kurzbeschreibung (.pdf)

Masterarbeiten

... nach Absprache Prof. Dario Anselmetti (D1-270) Kurzbeschreibung (.pdf)

Doktorarbeiten / Ph.D. Thesis

... nach Absprache Prof. Dario Anselmetti (D1-270) Kurzbeschreibung (.pdf)

Currently, we acknowledge funding from the following research grants:

   

  •  German Science Foundation (DFG) - DA 370/6-1: Molecular mechanisms of Desmin-ARVC-cardiomyopathy (2015-2017)
  • German Science Foundation (DFG) - DA 370/7-1: Translocation through biological nanopores (2015-2017)
  •   German Science Foundation (DFG) - DA 370/8-1: Dinuclear complexes that bind to DNA (2016-2019)
  • Industry project (Evonik): Nanoparticles and nanofluids (2016-2018)
  •  EU-Rocket Interreg Germany / Netherlands: Elasto-Tweezers (2017-2019)
  •   Forschungsfonds Medizin NRW/RUB/UniBi: Investigation of TMEM43-related cardiomyopathies (2017-2019)


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