Versatility and performance for biology and life science

For success in life science research, scientists depend on professional tools that can readily provide the information needed, regardless of the tasks at hand. By combining key technologies and components, Nanosurf has made the FlexAFM system one of the most versatile and flexible AFM systems ever, allowing a large variety of biological and life science applications to be handled with ease. With the FlexAFM, you can combine the liquid AFM imaging, spectroscopy and nanomanipulation capacity of this system with the high-end optical techniques available for inverted microscopes.

Flexible system design for life science research

FlexAFM comes with manual and motorized stages for seamless integration on Zeiss, Olympus, Nikon and Leica inverted microscopes or with standalone stages. On the inverted microscope, optical and AFM data can be correlated, as shown here for internal limiting membrane (ILM) of the human retina.

(A) Bright field image of isolated ILM in a physiological buffer. (B) Fluorescence image of the same section showing anti-laminin staining. (C) AFM topograph of a subsection of the ILM; also shown as overlay in B. (D) AFM stiffness measurements (stiffness map) of the same subsection. The color for each point represents the local stiffness value as calculated from force curves recorded at the respective positions. (E) Histogram of the stiffness data shown in D. (F) Typical force-displacement curves obtained on the ILM and on the glass substrate. These curves are converted to force-indentation data, which then allows calculation of the stiffness. Stiffness distribution of biological tissues has been shown to be a marker for diseases such as age-related macular degeneration, arthritis and cancer. Data courtesy: Marko Loparic, Marija Plodinec, Philip Oertle, and Paul B. Henrich, Biozentrum/SNI/UHBS, University of Basel, CH.

The modular stage, cantilever holder, and software concept allows an easy upgrade of the system to access many new possibilities in life science and materials research. Flex-FPM for cell and nano-manipulation, for example, and Flex-ANA for automatic nanomechanical analysis. In addition, advanced modes like MFM and KPFM that were originally developed for the Flex-Axiom system, are also available for FlexAFM. For measurements that don’t need optical access from below, e.g. for the imaging and spectroscopy of samples like bacteriorhodopsin, a standalone stage makes the FlexAFM compatible with the Nanosurf Isostage and Acoustic Enclosure 300, and generally makes the system much more compact.

A FlexAFM system with stand-alone stage, isostage, and acoustic enclosure. (B) 2D crystals of bacteriorhodopsin [140 nm scan range]. (C) Power spectrum of B, showing a lateral resolution of well over 1 nm [dashed circle]. (D) Single molecule force spectroscopy of bacteriorhodopsin.

FlexAFM lifescience application examples

Imaging of type I collagen fibrils

Collagen is the most abundant protein in mammals and contributes to more than 25% of the whole-body protein content. It is the main structural protein of the extracellular matrix of connective tissues and provides e.g. tendons and bone with their tensile strength. Most of the collagen found in mammals is fibrillar type I collagen. Type I collagen fibrils show a typical periodic morphology, the so-called D-banding. D-bands result from staggered self-assembly of individual collagen molecules into larger fibrils with a periodicity of about 67 nm.

Images of collagen fibrils from rat tendon were recorded in Prof. Snedeker's research group at the ETH Zürich. One of the reasearch areas of Prof. Snedeker is tendon mechanics and biology.

3D AFM topography of several type I collagen fibrils

AFM topography image of type I collagen fibrils

AFM deflection image recorded along with the topography image

The 3D representation of the AFM topography image nicely shows the typical periodic D-banding of type I collagen on all fibrils. The colloagen topography was recorded in static mode using a Nanosensors PPP-XYCONTR cantilever. AFM images were processed using Nanosurf Report Software. Preparation and imaging of collagen fibrils was performed by Massimo Bagnani, Prof. Snedeker research group, Uniklinik Balgrist, Institute for Biomechanics, ETH Zürich, Switzerland.

Measurements on living cultured cells

Mechanobiology is an emerging research area that deals with the effect of changing physical forces or changes in the mechanical properties of cells and tissues. Several diseases, such as fibrosis and atherosclerosis are associated with changes in tissue stiffness. Moreover, in cancer, the metastatic potential of cancer cells depends on their elastic modulus.

Here, the elastic modulus of living cells from a human breast basal epithelial cell line was measured using a Nanosurf FlexAFM system with the Flex-ANA software.

Elastic modulus map

Unperturbed cell topography from force mapping

The first image shows the elastic modulus (in kPa) recorded on living breast epithelial cells immersed in cell culture medium. Differences in the elastic modulus within the cell can be clearly observed. The dark area surrounding the cells originate from the much stiffer cell culture dish substrate.

The second image shows the unperturbed cell topography extracted from the force mapping data. The topography is determined from the contact point of each force curve and thus shows the cell topography at zero applied force.

Elastic modulus mapped to the 3D topography

Elstic modulus distribution

Mapping the elastic modulus data to the 3D topography allows relating the information of both channels. The 3D image was generated using Gwyddion software.

The last image shows the distribution of the elastic moduli extracted from nanomechanical force mapping experiments. The peak at lower moduli corresponds to the stiffness of the cells. The peak at the right originates from the cell culture substrate and shows much higher elastic moduli.

AFM data courtesy of Philipp Oertle, Biozentrum, University Basel.

Single molecule force spectroscopy of bacteriorhodopsin

The force-distance curve below reports the controlled C-terminal unfolding of a single bacteriorhodopsin (BR) membrane protein from its native environment, the purple membrane from Halobacterium salinarium.

Solid and dashed orange lines represent the WLC curves corresponding to the major and minor unfolding peaks observed upon unfolding BR, respectively. The contour length of the stretched polypeptides of the major unfolding peaks is given in amino acids (aa).

Single molecule force spectroscopy of bacteriorhodopsin

This data was recorded using a (10-µm; version 3) in combination with the C3000 controller and a cantilever with a nominal spring constant of 0.1 N/m (Uniqprobe, qp-CONT, Nanosensors).

FlexAFM image gallery

I型胶原纤维的AFM成像

细菌视紫红质细胞质侧的高分辨率成像

细菌视紫红质的单分子力谱

活体大鼠-2 细胞

DNA的AFM成像

组织样品

头发的动态模式AFM图

用AFM研究牙科植入物

蝴蝶翅膀的AFM图

The most flexible atomic force microscope for materials research

For success in materials research studies, scientists depend on professional tools that can readily provide the information needed, regardless of the tasks at hand. By advancing key technologies and designs, Nanosurf has made the FlexAFM one of the most versatile and flexible AFMs ever, allowing a large variety of materials research applications to be handled with ease. In combination with the powerful C3000i controller, complex material characterizations are possible.

The precision and performance you need for your research

The FlexAFM uses an extremely linear electromagnetic scanner for XY movement. This scanner delivers an average linearity deviation of less than 0.1% over the full scan range, top-ranking on the AFM market. The Z-axis is piezo-driven, with a position sensor that enables closed-loop operation. A sensitive cantilever detection system can measure well into the MHz frequency range. The scan head is connected to the full-featured, 24-bit C3000i controller with digital feedback and 2 dual-channel lock-in amplifiers.

Topography of SrTiO₃ in dynamic mode

Strontium titanate (SrTiO₃, STO) is an oxide of titanium and strontium exhibiting a perovskite structure. It has interesting and partly unique material properties. It is used as substrate for growth of oxide-based thin films and high-temperature superconductors.

STO forms surfaces that show a layered structure. The thickness of individual layers is in the range of a few Angstrom. Atomic force microscopy is an ideal tool to image and measure these structures.

Topography showing steps of strontium titanate; image size 1.1µm

Section profile and height distribution

The sample clearly shows the typical layer structure STO. Here, the layers are not perfectly smooth, but exhibit residual roughness of approx. 125 pm (RMS). This is caused by a non-ideal termination process during the preparation of this STO sample.

The graph shows the profile of the image shown above along a line extending from the top left to the bottom right corner of the imaged area. The profile also clearly shows the layered structure of the sample and reveals step heights of approx. 4 Å. Similarly in the right panel, the height distribution histogram of the image above clearly shows approx. 4 Å-spaced peaks for the different layers of the sample.

Topography and KPFM of CVD grown molybdenum disulfide monolayers

In this application note, monolayer MoS₂ grown by chemical vapor deposition (CVD) was imaged with Kelvin probe force microscopy (KPFM) using a FlexAFM to study the contact potential difference variation on a single crystal.

Monolayers of MoS₂ were grown on a silicon substrate by chemical vapor deposition (Sample courtesy: University of Illinois – Urbana-Champlain).

Non-uniformity of the contact potential signal across the monolayer can inform about doping profiles and other surface defects.

MoS₂ monolayers optical micrograph

a) AFM topography image of single MoS₂ monolayer. Location where profile is taken indicted by red line.
b) Height (top) and KPFM voltage (bottom) profile across monolayer

Measurements using the FlexAFM show a step height of 0.6 nm for the MoS₂ monolayer. Concurrent KPFM measurements show a 650 mV contact potential difference between the monolayer and the SiO₂ substrate.

3D AFM topography overlay MoS₂

All measurements were performed using a FlexAFM system equipped with a ANSCM-PA cantilever from AppNano. Images were processed using MountainsMap SPM.

For more information contact our application scientists

KPFM and MFM on stainless steel

In the experiment shown below, KPFM and topography data were recorded in a single run using a FlexAFM system. Also see the related .

KPFM overlay on a topography image of stainless steel
Scan size: 80 µm x 80 µm
Potential range: 200 mV

The topography image itself
Scan size: 80 µm x 80 µm
Height range: 50 nm

MFM image of the same area
Scan size: 80 µm x 80 µm
Phase range: 10°

FlexAFM image gallery

SrTiO3在动态模式下的形貌

棒状样品的AFM: 商业 Pt 电极上的 Cu 沉积

Pyrene纳米片的动态模式AFM

抛光不锈钢的磁力显微

抛光蓝宝石的动态模式AFM观测

聚合物混合物的AFM相图

用AFM对ePTFE膜进行形貌分析

飞机机翼涂层

牙科用抛光陶瓷片的接触模式AFM

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硬盘上比特的MFM

用AFM研究牙科植入物

不锈钢的静态力AFM

FluidFM®: The established microfluidic tool for single-cell biology and nanomanipulation

FluidFM literally brings microfluidics to the tip of an AFM cantilever. This way, it combines the microfluidics with the force sensitivity and positional accuracy of an AFM allowing a range of exciting applications in single-cell biology and nanoscience.

Nanosurf has a long experience providing AFM systems with FluidFM having launched the FlexAFM with FluidFM in 2011 together with Cytosurge. This integration of FluidFM on Nanosurf platforms has grown over time and now the FluidFM technology is available for Nanosurf's FlexAFM, CoreAFM and of course the flagship DriveAFM platform.

Learn more

A tool for research pioneers

• A tool to conduct original research at the frontiers of science from bacterial adhesion to cell-cell interaction and from spotting to injection and extraction
• A solution with optical, force, and fluidic control, with full software integration on DriveAFM
• FluidFM can also be combined with PicoBalance to measure the mass of cells and microparticles picked up by the FluidFM probe

Single cell adhesion, Colloidal spectroscopy, Single cell injection, Spotting, Single bacterium adhesion, Single cell isolation, Single cell extraction, Nanolithography, PicoBalance, SICM

FluidFM application examples with FlexAFM

Cell-cell adhesion forces

FluidFM cell adhesion measurements were extended to study cell-cell interaction. This can be the force between a cell (on the cantilever) and a cell below on a substrate (fig. 1 A), but also between a cell and its surrounding cells in a confluent layer (fig. 1 B).

Fig. 1: Cell-cell interactions (red springs) studied by aspiration of single cells to a hollow FluidFM™ probe (orange). A) Probing the force between a cell immobilized on the cantilever and a cell on the substrate, B) picking a single cell from a confluent layer, probing cell-substrate (purple) and cell-cell (red) interactions.

Dr. Noa Cohen of Prof. Tanya Konry's group at Northeastern university in Boston studied cell-cell adhesion forces with a Flex-FPM system to gain more insight into tumor progression and metastasis [Cohen et al. (2017)]. Figure 2 shows an optical image of the method depicted in fig. 2 A that was used by Cohen for this study.

Fig. 2: Optical images showing A) a single cell to be picked up by a FluidFM probe B) the cell aspired to the cantilever and C) the FluidFM probe with aspired cell during a cell-cell adhesion measurement.
Data courtesy of Tanya Konry group, Northeastern University, Boston, USA.

Single MCF7 breast cancer cell were immobilized on the cantilever. The cell was then pushed on different cell types that were immobilized on a substrate. The measured cell adhesion between MCF7 cancer cells to different cell types were found to develop differently with incubation time. In these experiments, the reversible binding of cells allowed the different cell pairs to be studied with the same probe (fig. 3), allowîng better comparison of measurements.

Fig. 3 A) Typical force curves between a MCF7 cell aspired to the cantilever and a non-cancerous, fibroblast (HS5) on the substrate at different contact times. B) Development of the force with contact time between the cells. Data courtesy of Tanya Konry group, Northeastern University, Boston, USA.

Dr. Ana Sancho from the group Prof. Jürgen Groll's group at the University of Würzburg extensively studied the interaction between a cell and its neighbors in a confluent layer of epithelial cells (fig. 2 B) [Sancho et al. (2017)]. Fig. 4 shows the cantilever picking up a cell from a confluent layer (A). After removal the empty space from where the cell was picked up is visible (B). Again, the epthelial cells of interest could be selected based on cell size and cell shape using the inverted microscope.

Fig. 4: Confluent layer of cells, where one is pulled out by FluidFM, adapted from: Sancho et al. (2017), Scientific Reports volume 7, 46152.

Human endothelial cells from the umbilical artery were found to exert strong intercellular forces (figures 6 A & B). The forces could be decreased significantly by overexpression of the so-called Muscle Segment Homeobox 1 (MSX1). The MSX1 induces a endothelial-to-mesenchymal transition. This transition is a process involved in cardiovascular development and disease. Complementary to these adhesion experiments, the Flex-FPM system was also used to perform nano-indentation measurements. To this end colloidal beads aspired to the cantilever and force curves were recorded on the cells.

Fig. 5: A) Typical single cell force curves of individual cells or cells in a confluent layer, depicting the increase in adhesion force by cell-cell interactions. B) Effect of MSX1 on the observed cell adhesion for individual cells and cells in a monolayer. Grey and black bars: control measurements on individual cells and monolayers, resp., pale and light blue MSX1 treated individual cells and cells in a monolayer, resp. Adapted from: Sancho et al. (2017), Scientific Reports volume 7, 46152.

Both examples strongly benefitted from FluidFM™ technology provided by the Flex-FPM solution. In case of the confluent layer the large forces of up to over 1.5µN eliminate chemical binding to study cell-cell adhesion forces. In both cases the reversible binding provided the experiments with the necessary speed-up to obtain sufficient statistics.

Spotting and lithography with FluidFM

In cell biology, micropatterning is becoming a widely accepted technique to address cellular behavior at the single-cell level. The ability to guide cell growth by local suppression or stimulation, for example, is beneficial for studying cell growth, for developing cell-based sensors, and for tissue engineering applications. The production of microfabricated biosensors is another application that requires precise placement of biomaterials on a substrate.

FluidFM® technology by Cytosurge enables deposition of (bio)molecules and particles at defined locations with micrometer accuracy and with femtoliter volumes. The closed channel is capable of depositing molecules from a liquid in both air and liquid environment. This enables numerous applications in biomedicine, cell- and microbiology, as well as non-biological nanolithography.

Automatically varying back pressure and contact time in a grid-like pattern (3 dots per condition), spot sizes can be quickly optimized.

Nanosurf logo written in in air with a solution containing approximately 50% glycerol; back pressure 200 mbar.

Innovative and intuitive handling

In the Nanosurf Studio software, FluidFM integrates seamlessly with the AFM operation software. Firstly, the pressure is directly controlled from the Studio interface where it can be synchronized with the spectroscopy. Secondly, ViewPort allows targeting of cells or areas of interest directly in live feed of the optical camera and running a queue of positions. Furthermore, with CleanDrive, DriveAFM uniquely provides the PicoBalance functionality to measure the mass of microparticles or single cells by FluidFM using a frequency sweep or PLL.

To learn more, also watch the FluidFM webinar presented by Wiley:

Download the FluidFM brochure for more information

ANA: The perfect tool for fully automated nanomechanical data acquisition and analysis

The ANA system is an automated solution for AFM-based nanomechanical analysis. It is designed to investigate the nanomechanical properties of materials such as cells, tissues, scaffolds, hydrogels, and polymers on multiple or large samples via force spectroscopy and force mapping in an intuitive and automated fashion.

ANA is compatible with Nanosurf's FlexAFM scan heads: for materials science, as well as for life science applications on a setup on an inverted microscope.

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I really enjoy using the Flex-ANA for its excellent force mapping capabilities on polymer materials. The scientists at Nanosurf are highly knowledgeable and responsive, and have developed an easy-to-use system with many features (both in hardware and software) purposefully designed for force curve measurements.

From automated calibrations and programmable stages to streamlined integration of multiple data sets, the Flex-ANA offers important benefits for any research involving force curve measurements.

Dr. Dalia Yablon
SurfaceChar

Nanomechanical analysis of large or multiple samples

The combination of a straight-forward workflow-based experimental procedure in the ANA software, carefully selected hardware elements and motorization, and well-designed and thought-through control algorithms are hallmarks of the ANA setup on FlexAFM. Measurement locations on large or multiple samples are defined by simply clicking on an overview image with a large field-of-view that covers the entire accessible 32 mm × 32 mm sample area. Height differences of up to 5 mm can be easily overcome by the system, and many types of nanomechanical measurements, analyses and models can be performed and used!

Handle demanding samples with ease

During an experiment, the ANA system coordinates the movement of the scan head and the motorized XYZ translation stage (range: 32 mm × 32 mm × 5 mm) to cope with large sample height variations. The system can optionally be equipped with an additional 100-µm Z piezo for an extended force spectroscopy range. Thus, samples with macroscopic and microscopic roughness in the range of several millimeters and micrometers, respectively, as well as soft and sticky samples, can be easily addressed.

Learn about details in our webinar by Dr. Edward Nelson

Learn about the automated nanomechancial analysis module and its application in analyzing blended polymers. See the ease of use in data collection and analysis, and how automated collection of data from multiple samples has the potential to save time and make your research more efficient.

Operation by experts as well as novice users

The ANA control software provides a Basic and an Expert user mode that are designed for different degrees of user experience and training:

In Basic mode, less experienced users are guided through the setup and calibration of the ANA system step-by-step. Optimized workflows guarantee that everything is done in the correct sequence, that nothing is forgotten, and that all is set properly.

In Expert mode, experienced users have more freedom in operating and setting up the system to suit their individual needs. Measurement parameters can also be prepared and stored by an expert, and then subsequently used by less experienced users in Basic mode.

The ANA software was partly developed within the framework of the OYSTER project, which has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 760827.

To learn more about ANA, please visit the ANA product page.

FlexAFM specifications and dimensions

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System specifications

Scan head dimensions