An Atomic Force Microscope is a device (Fig. A panel) that uses a cantilever (thin and narrow flexible plate) with a sharp or spherical tip in order to touch a sample. Touching of the sample allows the measurement of the mechanical properties of the sample. As the cantilever approaches and then indents the sample, the cantilever is first unaffected and later on bends as the force increases. This bending is precisely measured using a laser pointed to the cantilever, and measuring the deflection of this laser with a detector, as the cantilever bends (Fig. B panel) The force applied by the cantilever during the indentation, can be calculated in order to output the force-distance curves and the sample’s Young’s modulus, a measure of the stiffness, can be obtained. Another key features of the Atomic Force Microscope is the capacity to perform a very fast x-y scan of the surface of a sample to form an image from the mechanical information acquired. This image can be a 3D surface of the sample scanned by the AFM or a “stiffness” map of the sample based on the stiffness (young’s modulus) value measured at every pixel. Such image can range from the size of a whole tissue to the molecular and even close to atomic level. AFM thus allows a “mechanical” imaging of samples, providing invaluable information for our understanding of biological systems. The AFM can be used to probe the cell wall stiffness, the turgor pressure of a cells as well as for investigation of surface architecture.
Our AFM, NanoWizard® 4 XP BioScience with VortisTM2 Advanced version SPM Controller, HybridStageTM, DirectOverlayTM 2 software module and QITM- Advanced 2 software module, is equiped in high precision motorized stage and the associated software that allow both the automated screening of multiple samples placed on a same sample holder as well as the imaging of large samples by the “tilling” of adjacent scans of a same samples taken one after the other.
AFM is equiped with macroconfocalLeica LSI HSC. Macroscope of this system is equipped with an option of 2x/0.234 WD 39 mm or 5x/0.5 WD 19 mm macro objectives and a zoom function of 0.63 – 9.2x, alternatively a 63x/1.3 oil objective can be used. The macroscope has brightfield with Rottermann tilted illumination and fluorescence filter cube sets for DAPI, CFP, GFP, and RFP. This confocal has laser excitation lines at 405, 488, 561 and 635 nm and a one channel spectral detector where up to 8 sequential scans can be made for multi-imaging applications. The software of this confocal can be used for either single image acquisition (LAS AF software) or for custom made automated high content screening (LAS AF MATRIX), where parameters as multicolor imaging, 3D imaging, tile scans, time lapse studies, multiposition scanning, sample tracking, colocalization studies can be performed and it also has autofocus function. In addition, there are holders for 12x12 cm agar plates, microscope slides, multiwell plates, or small round Petri dishes.