The discovery scan is used to detect the fluorescence intensity, scattering intensity, turbidity, and position of each capillary along the length of the capillary tray. It is performed at the beginning of each experiment and the results are displayed in the Experiment Setup tab. Each capillary will be visible as a combination of two peaks, which represent the fluorescence intensity at 330 nm and 350 nm, respectively. In addition, the scattering signal from the DLS optics and the turbidity signal from the backreflection optics are displayed in a separate graph for each capillary (see image below).

After closing the drawer, PR.Panta Control performs an automatic discovery scan in Auto-Detection mode to determine optimal settings for an experiment. You can re-scan with different manual settings if necessary. Excitation power and DLS laser power can be set between 1 % and 100 %. The Prometheus Panta instrument determines the best settings based on the sample with the highest fluorescence and scattering intensity. PR.Panta Control will also detect the capillary format used ("tray type" single capillaries or capillary chips). The detected capillary tray type can be overwritten by the user next to the capillary selection.

Figure: Discovery scan results. Left: The upper plot shows both fluorescence wavelength peaks and the upper and lower detection limits (orange lines). The lower plot shows the scattering intensity from the DLS optics on the left y-axis (pink dots) and the turbidity signal on the right y-axis (green diamonds). Capillaries with non-aggregated samples usually exhibit a turbidity signal of 80-110 mAU, while maximum aggregation reaches values of up to 600 mAU. Right: In the capillary selection bar, capillaries with fluorescence intensities within the dynamic detection range are colored blue; capillaries with too high/too low fluorescence and/or to high scattering intensities are marked with an orange warning sign (upper graph). Left of the capillary selection, the detected capillary tray type is highlighted in dark blue (bottom graph).

The upper and lower limits of detection are highlighted by orange lines in the profiles. The upper limit is 20,000 fluorescence counts (Peak Fluorescence, meaning the height of the capillary peak) and 16,000 DLS scattering counts. Note that the lower detection limit is dynamically adjusted to the excitation power settings and thus may vary between experiments with different settings. Before continuing with a measurement, ensure intensities are within these limits. Please disregard the scattering intensity if you plan to perform measurements without DLS.

To zoom into the graph, use the scrolling function of the mouse wheel. Holding the mouse wheel allows the graph to be moved.

Note: The optimal detection range for fluorescence is between 2,000 and 18,000 counts. Some proteins might require limiting the maximal fluorescence counts to 15,000, since unfolding may result in an atypical increase in fluorescence intensity.

Note: Photobleaching effects are negligible even at high excitation intensities due to the rapid on-the-fly measurement mode.

Note: If the capillary fluorescence or scattering exceeds the upper limit, the capillary position will be indicated in orange and by default excluded from the measurement. Capillaries with fluorescence or scattering intensities below the measurement limit may yield data of poor quality but can be manually chosen in the capillary selection of the discovery scan.

Note: The fluorescence intensity is used to determine the optimal position for the DLS optics for each scan cycle. If the fluorescence intensity is below the measurement limit, a pre-programmed position will be used instead which may impair DLS data quality. Even if you plan to only measure DLS, make sure that the fluorescence intensity of the capillaries is sufficiently high.

Note: 100% DLS laser power is usually the most appropriate setting, because higher light intensity results in less noise. DLS laser power should only be reduced if the discovery scan shows detector saturation.