Sunday, 08 February 2015 14:18

Nanoscale texture of secondary cell walls

Plant dry matter or lignocellulosic biomass is a feedstock of bioenergy, biofuel production and novel biomaterials. Understanding the cell wall structure is important for effective utilization of the lignocellulosic biomass.

Structure, chemistry and mechanics of the polymer assembly of the secondary cell walls have been studied for decades on the macrolevel. However, characterization of the chemical components of the cell walls on the nanolevel are still unknown due to the limitation in spatial resolution of spectral techniques such as FTIR and Raman, which are used  for chemical analysis.  

Most secondary cell walls of xylem cells are made up of three dominating polymers: cellulose, lignin and hemicelluloses. Cellulose fibrils with diameter 3-4 nm are arranged in larger agglomerates with size of 20-25 nm and are embedded in a matrix consisting of lignin and hemicelluloses. Different theoretical models of special arrangement of the polymers in the cell walls have been suggested. Most of these models are based on the SEM or AFM study but not on the chemical information.

In this work, a Nanonics MultiView 2000 with Near-field Scanning Optical Microscopy (NSOM) was used  for the first time to study the photo-optical and thus chemical properties of cell walls at the nanoscale. This technique enables scientists to overcome the optical diffraction limit and to reach the spatial resolution of 50nm (limited to size of the NSOM aperture)

The cell walls of three different plants-beeches (hard-wood), spruce (soft wood) and bamboo (grass) were studied with NSOM.


Since the samples are semi-transparent, the NSOM measurements were conducted in reflection mode. In this mode the light coming out from the probe aperture interacts with the sample, is reflected and then collected from the top with an optical objective of an upright microscope. The NSOM measurements were performed with a Nanonics MultiView 2000 and Nanonics cantilevered NSOM probes. The MV-2000 scanning head has a completely open optical axis from the top and from the bottom and can be easily integrated with almost any kind of optical microscopes. MV-2000 together with cantilevered NSOM probes, which have extended tip and special geometry, enable true reflection NSOM measurements.

Three images were acquired simultaneously: height, phase and NSOM. The obtained NSOM images showed curls- like structures with dimensions about 125 nm and more, which are not seen in the height and phase images. It is most likely that lignin contributes a stronger NSOM signal than cellulose and hemicelluloses due to its interaction with light (autofluorescence and resonance effect). NSOM results obtained on the three different plants species (hard wood, soft wood and grasses) point to the universal principle of the special cellulose and lignin assembly in secondary cell walls. For the first time, NSOM images provide sub diffraction limited chemical information about the spatial distribution of the secondary cell wall components. This contributes to the understanding of the cell wall structure and its enzymatic degradation for energy conversion from ligninocellulosic raw material in general.  

Published:  Keplinger et al. Plant Methods 2014, 10:1 

Click here for more information on this Nanonics MV 2000 system

Published in Publication Highlights
Tuesday, 03 February 2015 20:13

Force spectroscopy on macrophages

A Multiview 1000 was used to probe the elasticity  of fixed macrophage cells with force spectroscopy.  Force spectroscopy was conducted on various cells with measurements performed around the nucleus.  A 3D image of a sample cell is shown below, with the force curves conducted at designated points on top of the cell.  Sneddon’s model was used to characterize the individual force curves to measure the elastic indentation of the cells; the Young’s modulus can then be calculated  by fitting the Hertz model to the data.  The authors find that the modulus of the macrophage cells increased significantly with adhesion to the ECM, showing that cellular adhesion plays a significant role in cytoskeleton and membrane softness.

Published:  Souza et al., European Biophysics Journal, 2014 DOI 10.1007/s00249-014-0988-3

Click here for more information on the Nanonics MultiView 1000 system

Published in Publication Highlights
Tuesday, 03 February 2015 19:33

TERS detection of membrane receptors

The ability to obtain chemical information about various receptors within cell membranes could advance efforts for drug development that target disease associated with receptor activity.  A key class of cell membrane proteins associated with cell adhesion, differentiation, and growth are integrin receptrs. In this work, TERS conducted with a Nanonics Multiview 4000 selectively detects the Raman spectrum of the integrin receptor in the cellular membrane.

Authors Hao Wang and Zachary Schultz of University of Notre Dame [ChemPhysChem 2014]use nanoparticles functionalized with ligands to bind with intact cell membranes.  The nanoparticle then functions as an enhancing nanostructure enabling TERS measurements of the Raman spectra of the ligands.  The authors conduct TERS to detect the signal of the amino acid within the integrin receptor and thus elucidate the structure of the receptor’s binding site.  This demonstrates the powerful utility of TERS for obtaining sensitive chemical information about amino acids relevant to chemical processes within cellular membrane proteins.

TERS setup for imaging nanoparticle functionalized ligands

Published:  H. Wang et al., 2014 ChemPhysChem   

Click here for more information on the Nanonics MultiView 4000 system

Published in Publication Highlights
Tuesday, 03 February 2015 19:33

AFM-Raman of nanoporous carbon from lignin

This paper by Spanish researchers Navarro-Suarez et al.  describes the development of a new technology to produce lignin-based nanoporous carbon with narrow and tunable pores. This novel material was prepared by chemical activation of natural lignin at 900˚C, and it was tested as an electrode material for electrical double-layer capacitors.

Characterization of the morphological, chemical and electrical properties of this new material was done by different methods including SEM, HRTEM, EDX, electrochemical analysis and Raman microscopy.  Raman characterization was performed with a Nanonics Multiview 2000 integrated with a Raman spectrometer. Such an integrated system enables characterization of morphological properties with simultaneous chemical characterization.  

The authors shows that there are ordered and disordered carbon regions and presence of few layer graphene (FLG) embedded into the amorphous carbon matrix.  The Raman spectral imaging and ratio between Iand IGRaman peaks of carbon confirmed SEM and HRTEM morphological results.  Further electrochemical studies showed that capacitance value of 6.87 Fg-1 can be obtained.

Thus, the obtained results demonstrated that nonporous carbon with tunable pore size can be achieved by using a natural material such as lignin and can be effectively used as electrodes material in energy storage applications. 

Distribution obtained by Raman spectral imaging of the ordered and disordered areas inside a particle for samples (a) 2, (b) 4 and (c) 6.

Color scale on the right varies with the ID/IG values from 0.0 (dark blue) to 1.2 (dark red).

Published:  RSC Advances, 2014, 4, 48336-48343

Click here for more information on the MultiView 2000 system



Published in Publication Highlights

Tuning forks for ultrasensitive force spectroscopy measurements

Link to PDF version of this app note


Standard AFM-based methods to monitor mechanical properties of materials such as elasticity and adhesion are based on beam bounce or optical beam deflection technology (see Figure below).  In this technology a laser beam bounces off the reflective back end of an appropriately chosen cantilever that is sufficiently soft to probe the material of interest  The laser beam then tracks the bending of the cantilever or its amplitude as a surface is being approached , which is then analyzed to understand the mechanical properties of the material of interest.

With such an approach there are two major problems seen diagrammatically below.  This schematic shows the tip-sample force as a function of time with two major sources of instability:  the jump or snap into contact resulting in a discontinuity, which happens upon the approach, and then a second discontinuity at the point of withdrawal resulting in uncontrolled ringing of the cantilever.  This is called Adhesion Ringing and is due to the fact that the adhesion force between the tip and sample is much stronger than the soft cantilever.  Thus, the adhesion of the sample in effect plucks the soft cantilever like the string of a guitar, resulting in uncontrolled ringing that has to die down.


A better controlled experiment would include a smooth approach and retract by the tip so that the interaction is described by a smooth mathematical function as indicated by the dotted line.

An improved approach to force spectroscopy:  tuning fork actuation and frequency based feedback

One way to avoid these discontinuity problems in the tip-sample interactions is to use the cantilever’s oscillation frequency instead of its amplitude as the feedback parameter.  Very stiff cantilevers with very high Q oscillations are typically used in such experiments.  The sharp frequency spectrum is very sensitive to small changes in the frequency imposed by ultrasmall forces of a tip interacting with a surface.  This method is called Frequency Modulation. 

An ideal example of a stiff device that is even self-oscillating (since it is formed of a piezo material) is a tuning fork, which also has very sharp resonance The higher the device Q the higher its sensitivity to any external perturbation such as surface forces. Thus, Frequency Modulation with tuning forks is ideal for force spectroscopy. 

This method has been implemented among varied research groups1 1b, 2and have validated the fundamental force limits of a tuning fork that were estimated to be less than a pN nearly a decade ago3 .  A Nanonics instrument recently demonstrated force sensitivity as predicted by Grober et al. in a beautiful study by Kohlgraf-Owens et al.4

These efforts have now been complemented by the pioneering theory of Sader and Jarvis5 that has shown theoretically that it is possible to derive accurate formulas for the force versus frequency in such Frequency Modulation methods.

Thus, tuning forks whose force constants are many orders of magnitude larger than standard silicon cantilevers have significant advantages in force spectroscopy measurements over conventional cantilevers.  By using such stiff tuning forks, the problems in classical silicon cantilevers of jump to contact and adhesion ringing are avoided.  Tuning forks provide high sensitivity especially in the region of very close tip-sample contact.  This region could not be addressed previously because of the discontinuities in the measurement but is now accessible..  Tuning fork feedback also provides other advantages, such as in electrical measurements, where in many semiconductor devices the feedback laser causes induction of carriers.  Finally, tuning fork feedback does not employ a laser so that there is no laser feedback interference for electrical and optical spectroscopy measurements.

Deriving model parameters from tuning fork based force spectroscopy

Tuning forks have force constants of 2,400 N/m and so their Q is much higher  than the tens or hundreds of N/m force constants of rectangular cantilevers.  The DMT model can be used to model force curves collected with tuning forks.

Finteraction is the force between the tip and sample

E* is the reduced elastic modulus

R is the tip radius

d0 is the surface rest position

Fadh is  an adhesion force

Discontinuities and hysteresis in the force curve can affect accurate measurement of the parameters that go into the DMT model.  A jump to contact instability prevents direct measurement of “do” and so the Finteraction between the tip and the sample will also be complicated.  Furthermore, if there is adhesion ringing then similarly the adhesion force can be difficult to assess. 

Determining the DMT model parameters using a tuning-fork based setup is straightforward.  With frequency used as the feedback parameter, amplitude can now be monitored as a separate independent quantity.  When a tuning fork with a tip approaches the surface with frequency feedback, the oscillation amplitude is measured independently.  Thus the point at which the tip touches the surface is observed immediately since one sees an amplitude change independent of any effect of feedback.  Furthermore, this occurs without any jump to contact so one knows the exact point of contact or “do” in the equation above. 

This is shown practically in the Amplitude vs Distance graph below.  As the probe approaches the surface from the right on the graph, from I, the point at which the slope abruptly changes, T, is the point of contact or “do”.  The intersection of the straight line I T and the straight line TM now define “do”.  M is the point of greatest penetration or indentation.  Similarly the point of retraction from the surface without ringing is readily and smoothly determined by the intersections of MR with the straight line RF.


The forces measured at T (FT) M (FM) and R (FR) are the Finteraction, 

Fmaximum or peak force and Fadhfrom Sader and Jarvis [J. E. Sader and S. P. Jarvis.5  We now have accurate formulas for the changes in Q, which is related to frequency, during the force distance curves.  Therefore, the forces at T, M and R can readily be determined even in ultra close proximity to the surface without any jump to contact or adhesion ringing and all elements of the DMT equation are known experimentally.  Note that the amplitude distance curves do not have to be symmetric.  They are only symmetric when there is no adhesion force.    

Nanonics in collaboration with the Continuum Mechanics and Material Theory Faculty at the Technical Univeristy of Berlin has tested the implementation of the equations of Sader and Jarvis.  These tests are based on extensive experiments with calibrated micromechanical suspended cantilevers with accurately determined force constants both theoretically and by other experimental methods.  An example of such measurements is shown below. 


The microbeams allow for the imposition of relatively large forces that enable direct comparison of the beam bounce method with theoretical results and with the tuning fork measurements. The solid curve above is a theoretical curve of the Force.  The measurement points are shown and the data compare well with the theoretical results.     

Now let us compare the tuning fork results on the same cantilever on the graph below.

The blue curve is obtained with the tip at the point where the microbeam is attached to the bulk silicon support where any bending detected is the result of the probe and can be used as a correction if needed.  The red curve is at the free end of the microbeam and the force is calculated with the Nanonics Software by measuring the frequency change and applying the Sader and Jarvis equations.  The yellow line is then generated by subtracting the extrapolated blue curve from the red curve; the blue curve can only be obtained at low force without breaking the tip since this curve is obtained at the inflexible point of the connection of the microbeam to the silicon support.  This removes any effect of probe bending which is in any case very small in this case with such a large force constant.

Now let us compare the slope of the yellow curve in the tuning fork measurements with what was measured by the beam bounce.  They are identical.  Thus, the Sader and Jarvis equations as implemented by the Nanonics program accurately determine the known force constant of these test microbeams with the curves crossing zero as would be predicted for 0 Force. 

Thus, today Nanonics is pleased to allow our customers a level of accurate force measurements with a variety of functional probes (optical, electrical, thermal, chemical deposition etc) that has till today been impossible to achieve by other techniques.


1.            (a) Ternes, M.; Lutz, C. P.; Hirjibehedin, C. F.; Giessibl, F. J.; Heinrich, A. J., The force needed to move an atom on a surface. Science 2008, 319 (5866), 1066; (b) Gross, L.; Mohn, F.; Moll, N.; Liljeroth, P.; Meyer, G., The Chemical structure of a molecule resolves by atomic force microscopy. Science 2009, 325.

2.            Albers, B. J., Three-dimensional imaging of short-range chemical forces with picometer resolution. Nature Nanotechnology 2009, 4, 307.

3.            Grober, R. D., Fundamental limits to force detection using quartz tuning forks. Review of Scientific Instruments 2000, 71, 2776.

4.            Kohlgraf-Owens, D. C., Mapping the mechanical action of light. Phys Review A 2011, 84, 011807R.

5.            Sader, J. E.; Jarvis, S. P., Accurate formulas for interaction force and energy in frequency modulation force spectroscopy. Applied Physics Letters 2004, 84, 1801.

Published in Application Notes
Monday, 12 January 2015 18:46

Biology Application



Applications in Bio SPM


Published in Application Notes
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