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Home > Teams > Dynamics of Complex Fluids and Morphogenesis > Tools: Experimental and Numerical approaches

Microscope à force atomique

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Introduction

The combined AFM/optical microscope at LIPhy

Some specifications of the setup

Contacts


INTRODUCTION

Principle of AFM operation: A probe at the extremity of the cantilever interacts with the sample under investigation. This interaction results in a vertical and lateral deflection of the cantilever of appropriate stiffness. This deflection is measured by the displacement of a reflected laser beam on a quadrant photodiode. Using calibration of the cantilever, the resulting differential signal is translated into a force and/or position information.
Atomic force microscopy permits to probe and manipulate a sample at the nanoscale by monitoring its interaction with a cantilever. Both the displacement of the cantilever and the force exerted on it can be measured very precisely by tracking the displacement of a laser spot reflected on its upper surface.



Cantilever and tip

The tip of the cantilever can be functionalized depending on the application : pyramidal tips are often used for probing the mechanical properties of the sample or obtaining the topology of the object. Colloidal probes are better suited for soft objects such as polymer brushes. These probes can be functionalized with various biomolecules to measure specific adhesion forces. More complex objects can also be attached to the cantilever such as entire cells.
Examples of tips: Left, colloidal probe of 5µm. Middle, pyramidal tip offering the best spatial resolution. Right, a cell has been directly attached to the cantilever for cell-cell interaction measurements.

Additionally, the cantilever itself must be chosen according to the application : low stiffness cantilever (0.01-0.06N/m) are commonly used for probing cells or soft substrates, whereas larger stiffnesses (0.5-50 N/m) are required for tapping mode experiments.


Measurement modes

Depending on the application, different measurement modes can be used :
Different operating modes: Left, example of force-distance curve mapping the interaction force between the probe and the sample as a function of the distance between them. Right, four different operating modes corresponding to different locations on this curve.
- Force spectroscopy using force-distance curves is used to probe locally the elastic properties of a sample or the interactions between the probe and the sample.

- Imaging modes (with or without contact of the probe) permit obtaining high resolution topological images of the sample. Visco-elastic properties of the sample can also be measured using the force modulation mode or the intermittent contact (or tapping) mode.


THE COMBINED AFM/OPTICAL MICROSCOPE AT LIPHY

The microscope combines an AFM setup with the ability to image the probe and sample under investigation. Transmitted light, phase contrast, RICM and fluorescence images are all possible thank to the inverted optical microscope on which the AFM head is placed.

An anti-vibration breadboard minimizes the artifacts for sensitive measurements. Temperature and perfusion controls are possible using dedicated sample holders.



A few examples of measurements performed with the setup.

Probing cell-cell adhesion: Left, A cancer cell attached to the cantilever is placed in contact with an endothelial monolayer. Right, the force distance retraction curve exhibits "jumps" of 20pN (at 0.5µm/s retraction speed) characteristic from ligand-receptor bonds.

The system is specially designed to be used for biology and soft matter experiments. Most experiments are performed in liquid phase in controlled environmental conditions.

Topography and elastic maps of biological cells.

SOME CHARACTERISTICS OF THE SETUP

- AFM : JPK Instruments Nanowizard II (Berlin, Germany) Equipped with CellHesion module (long range 100µm) and two sample holders for either petri dishes or coverslips (BioCell).

- Optical microscope : Zeiss Axiovision D1 (ph. Contrast / fluorescence) equipped with two sensitive CCD cameras (Hamamatsu ORCA-R2). A mercury lamp and two LEDs (470nm and 590nm) are available for fluorescence excitation. The microscope is equipped with 10x and 40x long distance air objectives and a high NA 63x oil objective.


CONTACTS

The "AFM-bio" is part of the Nanobio Platform that is accessible to researchers from and outside the LIPhy.

Get in touch with the AFM platform supervisors Claude Verdier or Valérie Laurent for additional information.

Technical assistance : Michaël Betton