BioScience AFM
Figure 2 : Setup of a tip-scanning AFM with an inverted optical microscope. The AFM head is placed on a motorized stage carrying a Petri dish holder for environmental temperature control and maintenance of living cells. Use of light optics is made possible by the transparent central part of the AFM head. The inset on the right depicts the basic optical lever principle of AFM detection in which cantilever defl ections are transferred to a four-quadrant photodetector. The AFM head (NanoWizard® 4a/JPK Instruments), is placed on an inverted light optical microscope (AxioObserver/Zeiss) equipped with a side-port fl uorescent camera (ProgRes®/Jenoptik).
transparent sample with condenser optics in an inverted optical microscope (IOM) is demonstrated in Figure 2 . T e choice of a tip scanner design is crucial for applications where the optical image of the specimen needs to remain in focus during AFM measurements. T e transparent central area of the AFM head is the only practical design that would allow the combination of top view optical access and simulta- neous AFM. T e piezo-scanner has a fl exure mechanism that decouples the xy -axes of motion from the z -scanner. T is is particularly important for high-resolution applications and data reproducibility.
Co-localization of all datasets . To make sure that structural and optical information can be truly correlated, it is important to remove any distortions arising from lens aberra- tions and non-linear alignment of mirrors in the optics system. Optical artifacts such as non-linear stretching, rotating, and off -setting of light microscope images are present in nearly all types of optical setups. By taking advantage of the accuracy of the closed-loop AFM, JPK’s DirectOverlay
to correct for any lens imperfections and transfer the optical image to the calibrated AFM coordinate system. An immediate benefi t is the possibility to carry out all measurements directly by choosing locations from within the optical image. An example is given with a super-resolution direct stochastic optical reconstruction microscopy (dSTORM) setup and Alexa-647-labeled microtubules in HeLa cells ( Figure 3 ).
™ feature is able 20
In addition to the accurate overlay of light optical and AFM information, the integration of both signals substantially reduces the total imaging time by avoid- ing the necessity of rescanning AFM areas, thereby reducing physi cal phenomena such as tip- contamination and the wearing of functionalized tips used for molec- ular recognition. Fast Scanning . T e application of conventional AFM imaging for imaging of soſt samples (particu- larly in liquid) is fundamentally limited to the use of very low im aging rates. T is is due to the rather slow feedback of the avail- able setups. Consequently, dynamic processes taking place at the sec- ond and even millisecond scale are impossible to capture. T ere is a range of developments made over the last two decades that make fast imaging possible. T e main improvements are a larger feed- back bandwidth, high-resonance z -scanners, and thermally quieter
cantilevers [ 4 , 6 , 10 ]. T e higher oscillation frequencies of the cantilever, larger feedback bandwidth, and enhanced xy- movement allow increased speed of the cantilever without decreasing the resolution of the measurement or damaging very sensitive samples.
distance curve based combined imaging and spectroscopy mode ensuring that not more than a set maximum force is applied to the sample ( Figures 1 c and 1 e) at each imaging location (pixel). Storing the entire force curve behind every pixel enables dedicated operations allowing extraction of quantitative data from QI images or maps. The QI software employs the acquired data to calculate the properties in question, using either already-available or a customized- fit algorithm. Typical parameters that can be extracted are work of adhesion, the actual adhesion, and contact point determination. Specific adhesion events can be detected, and Young’s modulus can be calculated using different contact mechanics models. Furthermore, the contact point height determination enables the possibility of creating the so called zero-force image (having no indentation), as well as examining the topography and indentation information from the sample at different forces within the range of the applied setpoint. This enables 3D tomographical reconstruction of the sample at a later stage and correlation of the information coming from different channels.
Quantitative Imaging™ (QI) . : QI curve is a force-
www.microscopy-today.com • 2015 November
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