Near-Field Scanning Optical Microscopy Simulation
In order to create a near-field image, the NSOM probe tip is scanned over the specimen with data collection occurring at defined intervals during scanning. This interactive tutorial explores the difference between scanning with the probe in feedback mode, in which the tip height varies in response to specimen topography, and scanning without feedback engaged.
When initialized, the tutorial begins with the tip engaged and scanning in Height Regulated mode over the specimen surface. In this mode, a signal derived from a feedback circuit regulates the separation of the tip and specimen. Selecting the Constant Height radio button switches off the feedback mechanism, and causes the tip to scan at a greater, fixed height above the specimen. While in constant-height mode, the tip of the probe must be positioned at least as high as the highest point on the specimen in order to avoid the two coming into contact, with possible damage to the tip and specimen. Note that when the feedback is turned off, collection of topographic data is not possible, and consequently the Topographical Image window on the right displays a constant signal (corresponding to a constant tip height). The Optical Image is produced in either of the scanning modes, although image quality is significantly better when feedback control is utilized.
The number of points collected in a given area depends upon the desired resolution, signal level, time constraints on image acquisition, and other factors. Acquiring a larger data set (greater number of data points) will result in a much longer scan time. A smaller data set will provide a shorter scan time with a resultant decrease in resolution. The data acquired during scanning is saved, processed, and displayed on a computer monitor. To engage the probe tip with the specimen, and maintain the engagement for meaningful data collection, some form of feedback mechanism must generally be utilized. Without feedback control of tip-to-specimen separation, either loss of signal intensity can occur (resulting in increased image noise) or the tip can be driven into the specimen surface.
In practice, scanning the probe in constant-height mode (no feedback) can be employed to determine if artifacts exist in the optical data due to the changing height of the probe. A cross correlation between the constant-height mode data and the height-regulated data will result in the most accurate optical dataset. In fluorescence localization experiments, tip height artifacts are not generally a problem, and the probe should always be kept in feedback mode to avoid damaging the tip.
The scan speed of the NSOM probe can be changed in the tutorial by adjusting the Scan Speed slider. Increasing the scan speed decreases the number of photons that can be collected at each point and therefore reduces the signal levels of the optical image. As the scan speed is increased past a certain point, the topographical image typically starts to deteriorate, due to the finite bandwidth of the feedback control system. As the scan speed approaches and passes the bandwidth of the control system, the feedback mechanisms can no longer respond with sufficient speed and the tip will occasionally run into the sample (assuming there are significant changes in the topography of the sample surface).