Using the Localization Progress Monitor

The Localization Progress Monitor displays an approximation of the final image reconstruction based on the current data collected. A fast calculation determines the positions of the fluorophores in each frame in real-time, or near real-time. The progress monitor is only an approximation of the final super-resolution reconstruction. The final data is fit to Gaussian functions in order to obtain a reconstruction with higher localization precision.

For each frame of the acquired data, the Progress Monitor first detects fluorophores with detection sensitivity n standard deviations above the mean of the raw image. The positions of the detected fluorophores are determined and plotted on the Progress Monitor display. The number of detected fluorophores for the current acquired frame is displayed in the upper-left corner of the Progress Monitor. The Progress Monitor is displayed as an image with 20nm pixels. The positions of each detected fluorophore are plotted as a single intensity count in the appropriate pixel, leading to a super-resolution display with localization precision equal to the pixel size (20nm in this case). Each time additional fluorophores are plotted in the image, the intensity count of those pixels increases, such that bright areas show the locations of where many fluorophores have been detected. If a sufficient amount of data has been collected, the user may elect to end the experiment early. While the experiment is running, the Progress Monitor can be cleared and restarted by clicking the Restart Monitor button from the Activation Power/Duration window.

To adjust the Progress Monitor settings:

1. Click on the More Run Options button on the Design/Run Experiment window. The More Run Options window is displayed.

2. Select the Show monitor check box to display the Localization Progress Monitor.

3. In the Skip frames before monitoring field, enter an integer number of frames to skip at the beginning of the acquisition before the Progress Monitor begins to plot data. This feature is designed to remove false positives produced from very densely labeled samples, where many of the initially detected fluorophores may not contribute to the final super-resolution reconstruction due to poor localization precision. Depending on the labeling density, it may not be required to skip any frames.

4. Enter the value n in the Monitor update frequency field to indicate that the Progress Monitor shall analyze every n frames of the experiment.

5. In the Monitor detection sensitivity field, enter and integer value, n, to determine the stringency for detecting a fluorophore. Fluorophores exhibiting counts that are n standard deviations above the mean intensity of the raw image are detected and plotted on the Progress Monitor.

Using the Localization Progress Monitor

1. Click on the More Run Options button on the Design/Run Experiment window. The More Run Options window is displayed.

2. Select the Show monitor check box to display the Localization Progress Monitor.

4. Enter the value n in the Monitor update frequency field to indicate that the Progress Monitor shall analyze every n frames of the experiment.

5. In the Monitor detection sensitivity field, enter and integer value, n, to determine the stringency for detecting a fluorophore. Fluorophores exhibiting counts that are n standard deviations above the mean intensity of the raw image are detected and plotted on the Progress Monitor.

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