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    Recognition Microscopy
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SCANNING PROBE RECOGNITION MICROSCOPY

Scanning Probe Recognition Microscopy is a new scanning probe microscope capability under development in the Electronic and Biological Nanostructures Laboratory (EBNL) at Michigan State University. In Scanning Probe Recognition Microscopy, the scanning probe microscope system itself is given the power to return to a specific nanoscale feature of interest through feature recognition coupled with adaptive scan plan generation and implementation. This recognition-driven and learning approach is made possible by combining scanning probe microscope piezoelectric implementation with on-line image processing and dynamically adaptive learning algorithms. This is an approach that works directly with the interaction sensing capability of a scanning probe microscope, which inherently has atomic to nanometer scale resolution. The human operator interaction is now focused to the decision-making level rather than the execution level, giving this approach tremendous potential for widespread high-throughput applications.

Figure 1. SPRM can scan along individual nanofibers in a tissue scaffold.
AFM	SPRM
	A coarse scan encountered the left hand nanofiber. A high resolution scan was initiated and performed to the end of the scan range. The tip was then returned to where coarse scan mode was interrupted and continued until it encountered the right hand nanofiber. See as SPRM video (.avi).

Figure 2. Adaptive SPRM enables high resolution scanning past the cross-over points.
AFM	SPRM
	Incorporation of adaptive learning in SPRM enables the high resolution scan along the nanofiber to continue past the cross-over point of two crossed fibers. See as SPRM video (.avi).

SPRM in Nanobiology

One of the most important research areas in which SPRM can have a significant impact is in nanobiology. Key issues in biology and medicine revolve around regulatory signaling cascades that are triggered through the interaction of specific nanoscale triggers with specific nanoscale receptor sites. This is exactly the scale which is amenable to investigation by scanning probe microscopy techniques. Direct cause and effect investigations require the ability to probe nanoscale sites in a reliable and repeatable manner. SPRM supplies this key enabling capability.

SPRM Investigation of Tissue Scaffolds for Spinal Cord Repair

The Michigan State University EBNL group collaborates with the University of Medicine & Density of New Jersey-Robert Wood Johnson Medical School group of Professor S. Meiners to investigate three-dimensional dense nets of polyamide nanofibers, which appear to have an unusually effective combination of properties for tissue engineering for spinal cord repair.

Figure 1. Sally Meiners, Ijaz Ahmed, Abdul S. Ponery, Nathan Amor, Virginia M. Ayres, Yuan Fan, Qian Chen and Ashwin N. Babu, "Engineering Electrospun Nanofibrillar Surfaces for Spinal Cord Repair: A Discussion," Polymer Int., Vol. 56, pp. 1340-1348 (2007)

Astrocytes cultured on even bare nanofibers adopted a stellate morphology (a) that is typical of their in vivo counterparts, whereas astrocytes cultured on poly-L-lysine (PLL) coated plastic coverslips had a flat, cobblestone appearance (b) which is never seen in the body. To date, PLL has been required for astrocyte growth in culture.

SPRM Investigations of Nanobiomedical Properties and Electrospinning Conditions

Using SPRM, we have performed the first investigations of surface roughness and elasticity properties directly along individual nanofibers. This was the first time that statistically meaningful information was collected along many individual nanofibers using an automatic procedure that maintained uniformity of experimental conditions. The SPRM approach provided a wealth of data. Our group has initiated the development of biologically meaningful metrics, and this work is continuing. In related work, correlations of electrospinning conditions with resultant nanofiber elasticity were investigated. Previous work had focused heavily on correlations of electrospinning conditions with nanofiber diameter. EBNL is the first group to focus on correlations of electrospinning conditions with the nanobiomechanical properties known to impact cell re-growth in nanofiber tissue scaffolds, such as surface roughness and elasticity.

Figure 2. From S.L. Rutledge, H.C. Shaw, L.L. Yowell, Q. Chen, B.W. Jacobs, S.P. Song and V.M. Ayres, "Self Assembly and Correlated Properties of Electrospun Carbon Nanofibers", Diamond and Relat. Mater., Vol. 15, pp. 1070-1074 (2006).

Atomic force microscopy (left) and Force Integration to Equal Limits work/elasticity maps (middle) of relative elasticity for poly (epri-caprolactone) nanofibers spun at bore radii (a) 152.4, (b) 254.0, and (c) 406.4 microns. Histograms (right) of the values indicate different mode (most frequent) values and also different distributions between the samples.

Figure 3. From S.L. Rutledge, H.C. Shaw, L.L. Yowell, Q. Chen, B.W. Jacobs, S.P. Song and V.M. Ayres, "Self Assembly and Correlated Properties of Electrospun Carbon Nanofibers", Diamond and Relat. Mater., Vol. 15, pp. 1070-1074 (2006).

Many force distance curves from data analyzed along several nanofibers have been averaged to produce the four mean-value force distance curves shown in the figure. The area under the force distance curve is the work done by the tip on the sample, which can be related to the inverse local elasticity E. The areas under the mean value force distance curves increased with increasing bore radius (insets), indicating a decrease in nanofiber elasticity (E) with increasing bore radius. The bore radius controls the initial droplet size and surface tension.