Scanning Probe Microscopy


Local scanning probe microscopy on nanostructured systems and biological materials

Scanning Probe Microscopy Scientific Activity

ISM-CNR is a leader institution in the field of Scanning Probe Microscopy. For more than twenty years, ISM researchers have designed, constructed and used Atomic Force (AFM), Scanning Near-field Optical (SNOM), and Scanning Tunneling (STM) Microscopes for material science and biology applications. This activity has also produced several patents and high-level scientific publications.

SNOM and AFM are well established techniques which, through the controlled scanning of a suitable sharp probe (a tapered optical fibre or a very sharp Si/Si3N4 tip respectively) above the sample surface, allow to collect topography images with resolution in the nanometer scale and, at the same time, to map other interesting properties of the samples. These “collateral” properties can be, typically, lateral friction/phase lag images when using AFM as well as reflectivity, transmittivity or fluorescence properties of the samples with a resolution well below the diffraction limit when employing a SNOM.Furthermore, both the techniques can be operated in air, in vacuum or in a suitable liquid environment (e.g. the physiological solution used to grow cells cultures). This latter, in particular, is an invaluable advantage when studying living systems.

All the instruments can be run in different modes of operations (contact, shear or tapping mode) and, in particular, the SNOM can employ different set-ups (tip illumination, tip collection or illumination/collection modes) optimized to match specific experimental demands.

Thanks to these peculiarities, the AFM and SNOM techniques have been applied in many research fields, spanning from biology to earth science, from the analysis of extraterrestrial samples to the traditional application in microelectronics and in the study of surfaces and interfaces for application in material science.

A brief description of the microscopes and some examples of applications are given below.

Imaging

Force Curves

 

Imaging

DNA-based nanobiosensors

dna_su_silicio

The development of biosensors, or in more general terms bio-devices, has recently become one of the major driving-forces of the rapidly growing biotechnology industry. This field includes a wide range of researches including, among other activities, genomics (study of an organism’s genome and the interrelationship between the genes), proteomics (large-scale study of proteins, particularly their structures and functions), computational biology, and pharmaceuticals.

In this work we have developed and optimized a protocol to prepare reproducible, fast and economic DNA-based biosensors on crystalline silicon substrates, identifying their limits and capabilities. Contact, Tapping and Phase Lag modalities of Atomic force microscopy have been exploited to characterize and optimize every step of the preparation.

Carboxylic acid terminated monolayer formation on crystalline silicon and silicon nitride surfaces. A surface coverage determination with a fluorescent probe in solution.

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, A. Mezzi and T. Prosperi.  Journal of material chemistry (2004) 14, 1461-1468

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidised crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridisation efficiency; parallel imaging of the process by Atomic Force Microscopy

F. Cattaruzza, A. Cricenti, A. Flamini, M. Girasole, G. Longo, T. Prosperi, G. Andreano, L. Cellai and E. Chirivino In press on Nucleic Acid Research (2006)

Application of spin-coating technique to produce ordered and controlled lipid bilayers

layer_lipidici

Dynamic mode atomic force microscopy images of DOPC sample deposited through spin-coating preparation. (a) The surface is covered by planes made by superposed unilamellar vesicles; (b) higher resolution image.

The reported picture regards a study of the morphological properties of multilayers made of cationic or neutral lipids (DOTAP and DOPC) and mixtures of them. The superposition of two layers is clearly visible in the top image and the features of the internal layer are stressed in the second one.

Samples were obtained with a recently proposed technique of lipid film deposition, in which stacks of a small and well-controlled number of bilayers are prepared using a spin-coater. We optimized the technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate and disclosed the possibility to control the lipid’s structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.

AFM characterization of solid-supported lipid multilayers prepared by Spin-coating

G. Pompeo, M. Girasole, A. Cricenti, et al.  Biochem Biophys Acta – Biomembranes, (2005) 1712(1), 29-36

Induction of apoptosis in granulocytes

apoptosi

Lateral force (a,d), topography (b,e) and cross sections (c,f) relative to different granulocytes bearing lateral frictions contrasts on their plasma membrane

Induction of apoptosis in human granulocytes due to withdrawal of an essential growth factor (Interleukin-3) have been observed and characterized by AFM. Beyond the pure observation of the morphological path, the use of the AFM allowed to describe the occurrence of lateral friction contrasts associated to chemical/mechanical alterations of the plasma membrane of cells incoming the apoptotic pathway. No effects have been observed on control cells nor on necrotic cells suggesting that the membrane alteration is a well defined and never previously observed phenomenon triggering the latest step of the apoptosis pathway.

Atomic Force Microscopy Detect Transient Frictional Contrasts in Apoptotic Cells Induced by Deprivation of Interleukin-3.

M. Girasole A. Cricenti, R. Generosi, et al.  Appl. Phys. Lett. (2001), 78(8) 1143-1145

 

Photonic crystals: LiF-based nanoradiographies

LiF_samples

LiF is a well-known radiation-sensitive material, that can be grown as a thin film on different substrates.

LiF was used as image detector for X-ray micro-radiography and X-ray microscopy exploiting the fact that extreme ultraviolet (EUV) radiation and soft X-rays can produce, in LiF, stable point defects, known as color centers (CCs). Thus, LiF crystals and thin films can be successfully used as image detectors in soft X-ray contact microscopy. Biological samples can be placed in contact with the LiF film, exposed to EUV and soft X-rays (20eV – 1500eV) produced by a laser-plasma source and then removed.

Within the various kind of optical techniques, Scanning Near-field Optical Microscopy (SNOM) offers the possibility to investigate the optical properties of this kind of samples with a resolution in the nanometer range.

With this technique, we have studied several samples, in particular we have performed micro-radiographs of a mosquito (Diptera) wing, stored in a 120 nm thick LiF film evaporated on a silicon substrate.

 

Studies of Red Blood Cells

  rbcmod1d

AFM images showing pathologically occurring shape and artificially induced modification in RBCs. Panel (a): anisopoichilocytosis; (b): normal cells incubated with 2 mg/mL lecithin; (c): normal cells incubated with 10 mg/mL lecithin. Panel d: spherocytosis; (e): normal cells after incubation in hypotonic (210 mOsm) buffer, a ghost is also visible in the image; (f): normal cell after incubation in hypotonic buffer followed by treatment with 200 mmol chlorpromazine.

The occurrence of unusual shapes in pathological RBCs can be simulated by artificial treatments of normal cells with suitable chemical or physical agents. Such an approach can be useful to understand the mechanism underlying some cell alterations, especially in the classes of pathologies whose primary molecular defects is unknown.

A more detailed approach to characterize normal and pathological differences in RBCs, based on the systematic study of the membrane roughness of RBC has recently been developed. Accumulating data shed more light on the capability to really simulate a pathological RBCs by means of an artificial treatment. More remarkably, the possibility to discriminate between normal and membrane-skeleton defective (pathological) cells on the base of the behaviour of novel morphometric parameters resulted as well.

Such a novel approach can also be applied to the study of the natural or artificially-induced aging of the RBCs. Indeed, preliminary data demonstrate that the roughness of the plasma membrane can give precious information on the progressive cell aging (paper in preparation) which, in the future development of the study, has to be correlated to the formation of hemicromes and Heinz bodies in the interior of the erythrocytes.

Atomic force microscopy study of erythrocyte shape and membrane structure after treatment with dihydropiridinic drug.

M. Girasole, A. Cricenti, R. Generosi et al.  Appl. Phys. Lett (2000) 76, 3650-3652

Artificially Induced Unusual Shape of Erythrocytes: An Atomic Force Microscopy Study.

M. Girasole M.,A. Cricenti, R. Generosi, et al.  J. Microscopy. (2001), 204) 46-52.

Roughness of the Plasma Membrane as an Independent Morphological Parameter to Study RBCs: a Quantitative Atomic Force Microscopy Investigation

M. Girasole, G. Pompeo, A. Cricenti, A. Congiu-Castellano, F. Andreola, A. Serafino, B. H. Frazer, G. Boumis and G. Amiconi

Biochem. Biophys. Acta – Biomembranes.  Feb/2007

DOI link: http://dx.doi.org/10.1016/j.bbamem.2007.01.014

 

Interaction of electromagnetic fields with biological systems

 

fig_CM_colore
cross section CM

Human lymphoblastoid cells exposed to 50 Hz, 2mT magnetic field for 9 (B), 36 (C) and 64 h (D). A control cell (A) is also reported. The morphological alteration detected as consequence of the exposure has been exemplified by the trend of the reported cross sections.

 

Electric and/or magnetic field interact with biological systems producing consequence that can be either dangerous or health-saving depending on several parameters of the field (intensity, exposure time, frequency, wave shape etc.) or peculiarities of the biosystem.

We used a sinusoidal, 50 Hz, 2 mT, magnetic field which has been continuously delivered to human lymphoblasts up to 64 hours. The exposure to the magnetic field resulted in morphological modification of the human lymphoblasts. The characterisation of the membrane surface reveals an initial loss of microvilli followed, at longer exposure, by the progressive appearance of ripples, furrows and membrane infolding. Also the overall shape of the cells changes at increasing exposure as they become wider and less domed, an effect which has been interpreted as consequence of a magnetic field driven -and calcium induced- disgregation of the cell’s cytoskeleton.

Modifications of Human Lymphoblastoid Cells Induced by Low Frequency Magnetic Field: a Three-Dimensional Atomic Force Microscopy Study.

M. Girasole, A. Cricenti, R. Generosi et al.  Appl. Phys. Lett. (2001), 78(20), 3145-3147.

 

Following bacterial and cellular growth

colicoli

A bacterial colony growing on a neuron network.

In the above reported image, the growth of a bacterial colony contaminating (at room T, and in ambient conditions) a neuron network is shown. This images exemplifies one of the most intriguing application of the modern SPM techniques, which is to provide the opportunity to follow the evolution of a dynamical process.Such a capability, which can be exploited to study both organic and inorganic samples, makes SPM a unique tool for “ex vivo” investigations and justifies the increasing prestige of these techniques for applications in the biology-related fields.

incubatore

The implementation of a bimorph-based Tapping-SNOM with a Binder CB150 incubator allows to follow the dynamical evolution of more delicate biological systems which require a more strict control of the environmental conditions (temperature =37 °C, and 5% addition of CO2).

In this controlled environment the evolution of several different cell lines, all chosen with the common characteristic of growing in adhesion on a Petri cell, have been successfully attempted. The reported images, in particular, refer respectively to PIg Endothelial Cells (PIEC), RBC, and LAN-5 cells.

living_tap

Topography and phase-lag images of PIEC cells. The phase-lag, in particular, reveals clear cell-cell and cell-substrate contrasts

rbc_resolution

Topography (left) and 630nm-reflectivity image (middle) taken in the white square drawn on the topography of a rehydrated RBC in PBS buffer. A remarkable, sub 100-nm resolution is also shown (right)

filopodia

3D topographies, collected shortly after one another, of the very same LAN5 cell. The images show evidence of cell motion both for the overall shape rearrangement of the cell and for the presence, in the right image, of pseudopodia-like filaments at the two sides of the cell.

An alternative tapping SNOM setup enabling the study of biological systems in liquid environment

M. Girasole, G. Longo and A. Cricenti. J.J.A.P. 2006; 45: 2333-2336

 

Characterisation of nanoparticles in extraterrestrial materials

imm_homepage

AFM topography of chromite in a meteorite

The association between the most abundant population of meteorites, the ordinary chondrites, and their parent bodies is one of the main topics in the quest to understand the evolution of the solar system. This association is mainly inferred from spectra in visible and near-infrared wavelengths, where many of the asteroids show reddened reflected light curves. The analysis of lunar soils attributed the reddening of the spectra to the presence of nanometer-size metallic particles, and a simulation of micrometeoroid impacts with laser irradiation experiments on terrestrial samples has reproduced this effect and tied it to the vaporization of Fe-bearing silicates. Recently, spacecraft observations have revealed a new mystery in the interpretation of the reddening of S-type asteroids.

We have identified an alternative process for surface alteration of airless bodies that can be invoked to solve this mystery through a shock-induced phase transformation of Fe-Ni alloys. These metal phases are usually reported in meteorites but have never been associated with the reddening of the spectra. Here we show, for the first time, atomic force microscopy observations of diffuse nanostructured metal in ordinary chondrites. We also show that the corresponding spectra are unambiguously redder.

Discovery of nanophase metal in meteorites: implications for space weathering of asteroids.

P.F. Moretti, A. Maras, E. Palomba, M. Girasole, G. Pompeo, G. Longo, A. Cricenti, F. Somma, L. Colangeli and M. Serracino. Astrophysical Journal Letters  634, L117-120 (2005)

The present research line, namely, the study of metal nanoparticles has also be extended to the characterization of their formation mechanism in ordinary steels. In particular the characteristics and the penetration depth of the nanoparticles as consequence of mechanical solicitation have been investigated.

The present study has also produced a patent (“A new tool to detect the hardening of metals or metal-containing materials through the analysis of the VIS/NIR spectrum”)

AFM characterization of air blast shot peened stainless steel: nanocrystallization diagnostic for industrial purposes

G. Pompeo, M. Girasole, G. Longo, A. Cricenti, A. Maras, M. Serracino and P.F. Moretti – Submitted to  J. of Microscopy

 

Free Electron Laser (FEL) applications in the IR range

Application in the field of biology and material science can take great advantages in term of chemical sensitivity, which is the key to provide a localization of selected structures or components, by the implementation of the standard SNOM techniques with the Near InfraRed (NIR) radiation from a Free Electron Laser (FEL).

For instance the selective absorption of a cell at the wavelength of the amide-1 and 2 as well as the chemical sensitivity on diamond clusters are shown as few examples of the many possible applications

FEL_pancreatiche

FEL_diamond

For more detailed information, please, see the following links:

http://www.vanderbilt.edu/about.html

http://www.lightsources.org/cms/?pid=1000447

Other fields of interest:

HEAVY METAL POLLUTION and NANOTOXICITY

In last years both scientific results and epidemiological reports have suggested many questions regarding the possible negative effect on public health of the increasing environmental pollution, especially concerning the presence of heavy metals and metal-containing nanoparticles.

While somministration of heavy metal as metal salts dissolved in a buffer solution is known as a possible inductor of cell death by either triggering apoptosis or necrosis, the mechanism of action of metal nanoparticles is less clear.

In the past years we studied the cellular mechanisms triggered by the addition of three different metals: cadmium, lead and zinc. All of them have been administered to pancreatic (INS-1) cells, as chloride salts at a concentration of 100 µM. The AFM and LFM results allowed to discriminate between the different mechanism of cell death induced, on one hand, by Cd and Zn and, on the other hand, by Pb. The major effects have been observed in term of overall cell’s shape alteration and, more interestingly, on the occurrence of lateral friction contrasts on the plasma membrane which can be related to chemical modification induced on the cell’s surface.

Concerning the interaction between living systems and nanoparticles, we are currently developing an experimental approach devoted to the study of nanoparticle’s accumulation and internalization in cultured cell lines. On the base of our previous results on Me-Cl2 somministration, we are interested in the investigation of the different biochemical mechanisms triggered by Cd, Zn or Pd-based nanoparticles. The effect on different cell lines will be correlated, in a first approach, to the size of the selected nanoparticles.

fig1_girasole              fig3_girasole

Doping of pancreatic INS-1 cells with Cd, Zn or Pb induce different classed of morphological modification of the overall cell’s shape (left images: from top to down control, Cd, Zn and Pb-doped cells). On the surface of Cd and Zn-doped cells, but not on controls neither on Pb-doped cells, the occurrence of lateral friction contrast have been observed.

 

Force Curves

The AFM technique has been originally developed as a pure imaging technique. Subsequently, however, its capability to detect and measure the forces between the sharp probe and the sample surface became a very attractive tool disclosing the possibility to perform a number of unique experiments in the fields of material science as well as biophysics. Such studies are based on the collection of the force curves which are plots of the cantilever deflection (i.e. force detected by the tip) versus the tip-sample separation (force-distance curves). The ideal behaviour of such curves is sketched in the following figure.

curva forza

Ideal sketch of the tip-sample forces. When the probe is far-away from the sample, the cantilever is in its rest position and no force is detected (A). Approaching the tip to the surface, the cantilever bends towards the sample under the effect of attractive forces, driven essentially by Van der Waals’ (B). When the attractive gradient overcomes the cantilever elastic response, the tip plunges to the sample surface (jump to contact). After that, the tip is in contact with the sample and the cantilever is deflected by a repulsive regime of interactions (C).

In the everyday laboratory practice, the force curves are acquired by collecting the cantilever deflection while the tip is driven closer to the sample surface (approach curve), and when the tip is subsequently withdrawn (retraction curve), through the elongation/retraction of a piezoelectric scanner. Since the cantilever exhibits a typical elastic response, the deflection can be easily converted into force measurement by exploiting the relation: F=-keffΔx where Δx is the deflection and keff the elastic constant of the probe-sample system. This procedure, however, allows to collect a force vs scanner displacement curve rather than a force versus actual tip-sample distance. The difference between distance and deflection is depicted below.

 

distance_displacement

Cantilever deflection versus tip-sample distance

Thus, before the information about the sample properties can be obtained, the force vs. displacement curve must be analyzed and converted into a real force versus distance curve by taking into account the cantilever properties and the effects of the force experienced by the tip during the data collection.
The differences between the above described curves are reported below.

steel_force_vs_displacement
steel_force_vs_distance

A typical conversion from force vs displacement to force vs distance curve. Data are collected on a stainless steel surface, a useful reference sample. Indeed, exploiting the linear behaviour in the contact line (non-deformation hypothesis), this surface can be used to calibrate the force axis, that is, to determine the photo-detector sensitivity (in mV).

Examples and applications

In the following, examples of force versus displacement curves are shown. All the experiments are performed with an home built AFM equipped with a liquid cell in order to study the surfaces both in air and in suitable liquid environment.

 

mica_in_air
mica

(a): Approach and withdrawal curves on a mica surface in air. (b): The structure of a vertical slice through freshly cleaved muscovite mica.

 

 

Data shown in fig. a above are useful to introduce the differences between approach and withdrawal curves. The main differences regard the contact line (1) and the position of the jump-off contact point (2). Several factors participate to the appearance of the variation in the contact lines, first of all the scan velocity and the non linear response of the piezo transducer. Concerning the jump off contact, it occurs at larger pulling (negative) forces and this is the consequence of the meniscus interaction (wetting layer) between the tip and a thin layer of water adsorbed onto the surface (experimental conditions: air, 30 % relative humidity).

The use of a liquid cell allows to perform more sophisticated experiments. Namely, it allows to remove the meniscus force and study the relevance of other phenomena (primarily double layer and Van der Waals forces).
For instance, the force curves collected on the very same mica sample in liquid at different pH in the range 5 to 8 (and remarkably low ionic strength: 30-50 μM) are reported in the following figure.

mica_ph_a mica_ph_b

Force curves acquired on mica surface in liquid at various pH values. Curves are artificially shifted to evidence the differences. (a) Approach curves: it is worth nothing the disappearance, for high pH values, of the attractive regime area. (b) Withdrawal curves: the pH modulate the jump off contact amplitude

 

Comparing the withdrawal curves in air and in liquid (compare the curves in the b side of this figure with the red curve of the previous one), a strong reduction of the pull-off distance can be seen. Moreover, the chosen pH range, while far from extreme values, is large enough to allow the observation of a trend in the forces behaviour: at increasing pH values, we observe the gradual disappearance of the attractive regime (approach curves) and a strong reduction of the amplitude of the jump off contact (withdrawal curves). The disappearance of the attractive regime in our experimental conditions is due to the balance between the attractive Van der Waals forces and the double-layer forces produced by the charges (resident ions and surface dissociation) in the liquid.

More theoretical and experimental information can be found in
Butt, H. J. et al., Surface Science Reports 59 (2005), 1-152