Microtubules of living human skin fibroblasts are manipulated with the NanoWizard® Atomic Force Microscope (AFM) and imaged with Stimulated Emission Depletion (STED) microscopy. The Microtubules were labeled with SiR Tubulin and imaged with an Abberior Instruments Expert Line STED microscope (excitation, 640 nm; STED, 775 nm). The AFM manipulation path is marked with an arrow, the manipulation force was set to 5 nN with a velocity of 1 µm/s.
The bending and manoeuvering of the microtubules by the AFM tip can clearly be monitored by the STED microscope in real-time. Microtubules can be targeted and manipulated on the nanometer scale via the JPK DirectOverlay™ feature. The pressure exerted by the AFM tip can be monitored and controlled. This can provide more information about the elastic behavior of microtubules.
Abberior Instruments GmbH
Living A549 cells were measured at 37°C using a NanoWizard BioScience AFM. Microtubules were labeled with silicon rhodamine (SiR-tubulin).
Sample courtesy of:
Dr. Josef Madl, Prof. Winfried Römer
AFM and confocal fluorescence images of a phase-separated lipid bilayer sample. The background liquid disordered phase is around 1 nm lower than the liquid ordered islands. DirectOverlay™ was used with the NanoWizard BioScience AFM. Scan field: 15 µm x 15 µm; Z-range 2 nm.
Sample courtesy of:
Dr. S. Chiantia, group of Prof. Schwille
AFM and confocal fluorescence images of a confluent monolayer of MDCK cells with labeled actin. The rough texture on the surface comes from the characteristic microvilli covering the apical surface. Imaged with a NanoWizard BioScience AFM with DirectOverlay™.
Simultaneous AFM and FLIM measurements on nanoparticles. Scan size 800 nm². Imaged with a NanoWizard NanoScience AFM.
AFM and confocal fluorescence images of two SAOS cells in buffer. Actin fibres were labeled with FITC-phalloidi. A NanoWIzard Sense+ AFM with DirectOverlay™ was used. Scan field: 75 µm * 100 µm; z-range: 10 µm.
This movie demonstrates the seamless integration of confocal scanning with optical particle manipulation as well as the multiple degrees of freedom offered by the NanoTracker™ system in positioning a sample relative to a trapped object.
A fluorescent bead is optically trapped and moved in 3D through a sample of fluorescently labeled cells. Movements of the cell sample in x, y, and z are performed using a three-axis piezo scanner (when the cells move relative to the field of view or the focus changes). For movements of the bead relative to the camera image, the NanoTracker's 3D trap steering system is used (when the bead moves in x and y or in and out of focus). The movie nicely shows that the sample can be positioned in 3D relative to a trapped bead and also the bead can be freely moved through the stationary sample.
AFM and fluorescence images of p-Hexaphenyl polymer nanofibers formed on mica. For perfect integration, the DirectOverlay™ feature of the NanoWizard NanoScience AFM was used. Scan field: 8 µm x 8 µm; z-range: 60 nm.
Dr. F. Balzer
AFM and fluorescence image of Alexa555-labeled Rad51 proteins bound to DNA. Imaged with a NanoWizard BioScience AFM.
Rad51 assembles into filaments along double-stranded DNA, which can be seen in the 3D plot of the 700nm topography image. In the fluorescence overview image, the Rad51 filaments are red fluorescent, with the AFM scan regions superimposed. Each blue AFM scan encloses a single DNA molecule, partially coated with Rad51. To precisely and easily combine AFM and optical microscopy, DirectOverlay™ was used.
An overlay of a 3D contact mode AFM image (in red) with the corresponding, calibrated optical image (yellow-green fluorescence) using the DirectOverlay™ feature of a NanoWizard BioScience AFM. Living REF52 fibroblasts expressing YFP-paxillin. Fluorescent imaging with 63x oil immersion lens. Scan field: 100 µm x 100 µm; z-range: 3 µm.
This video playfully demonstrates the versatile manipulation of single microtubules using the NanoTracker™ optical tweezers instrument in conjunction with simultaneous sensitive fluorescence microscopy. The microtubules are fluorescently tagged using a chemical dye. Two optical traps are used to hold and manipulate the microtubule from both ends. Such constructs are key in biochemical / biophysics assays that probe the mechanics of microtubules, or of so-called motor proteins that bind to microtubules and walk along them by converting chemical energy into mechanical energy.
Many such experiments have revealed the intricate function of such motor proteins, allowing researchers to both understand such motor proteins as they operate in their cellular environment, and how chemical drugs can affect their functionality, opening the path towards more biomedical applications.
2-5: Zoom into region scanned with AFM showing 100 μm×100 μm scan (height range 5 μm) and inset 15μm×15μm (height range 2 μm) scan topography images using PeakForce Tapping. The feedback correction signal images highlight the surface membrane features, particularly in the zoomed image. Microvilli dominate the center of the cell, with membrane ruffles at the cell boundary.
AFM topography, adhesion, elasticity, and DIC images of living dorsal root ganglion cells, imaged with a NanoWIzard BioScience AFM in QI™ mode. For perfect optical integration, DirectOverlay™ was used.
Living CHO on gold electrode measured in PetriDishHeater™ for BioMAT™ at 37 °C. Cells were fluorescently labeled with Hoechst (blue nuclei) and fluorescein diacetate (green cytoplasm) and imaged with 40x dipping objective and AFM QI™ mode.
Images of isolated acidithiobacillus bacteria grown on the surface of pyrite (an iron sulphide mineral). The mineral substrate is opaque, so optical images were taken using an upright microscope with the BioMAT™ stage to allow fluorescence imaging in the same location as the AFM images. Scan region: 13 µm * 13 µm; Z-range: 410 nm.
Images courtesy of:
S. Mangold, group of W. Sand
University of Duisburg
Force mapping height and Young´s modulus image of fixed mouse cerebellum tissue. The BioMAT™ Workstation was used to overlay a 63× upright fluorescent microscopy image of DAPI stained nucleus and AFM force mapping height. Newly developed automatic height compensation was used to overcome the typical large height differences of the tissue sample.
Sample courtesy of:
AG Prof. Jochen Guck
Dr. Elke Ulbricht
TU Dresden, Germany