Load SBDx

This button opens a standard Windows Open File dialog allowing the user to open a batch job file.

Save SBDx

This button opens a standard Windows Save File dialog allowing the user to save the batch job file into the specified folder with a specified name.

Add current ND2 as task

This button inserts a new task line. The task line is populated with an ND2 file path, ID parameters, and selected period range currently set in the main dialog.

Delete selected task(s)

Deletes the selected tasks.

Delete all the task(s)

Deletes all tasks.

Move selected task(s) up

Moves the selected task(s) up.

Move selected task(s) down

Moves the selected task(s) down.

Edit Identification parameters for selected task(s)

Opens the Identification Settings dialog used for this input (please see Identification Settings).

Start Batch Analysis

Executes the batch job. Once the job is executed, this button changes to Terminate Batch Job so that the batch job can be stopped.

Input

Only the selected file type can be added to the list of tasks.

Default Text Output

Sets the default text output.

Each row corresponds to one task. If this option is checked, the selected task is included in the current job.

Data Set

Shows the file system path to the raw data set associated with each task. Double-clicking on the data set name opens the selected source image.

Identification Parameters

Shows identification parameters for each task. Double-click on the parameters opens the Identification Settings dialog window used for adjusting the parameters (please see Identification Settings).

Analysis Range

Displays the period (or Time slice for Live Cell data) range for each task.

ROIs

Displays any available ROI(s).

Text Output

Specifies the optional text output format.

Status

This column is active only when the batch job is running. It displays the status of each task. For the tasks that have not yet started it is blank. For the currently active task it displays the percentage progress of each step. For processed tasks the status can be “Completed” or “Terminated” or “Failed” for STORM Analysis tasks, or number of molecules output for Molecule List Conversion tasks. Some of the common failures have specific descriptions such as “Failed to load” or “Failed to Identify”, etc.

unique/overwrite/rename old

Unique (default) generates unique names as described above. Overwrite saves the output with a default name, overwrites the old one if it exists. Rename old saves the output with a default name, renames the old one if it exists.

Source

Set the source area to be exported either by entering the Top/Left/Width/Height values or simply by repositioning the red rectangle in the image. Make sure proper units are selected.

Target

Set the target size (Width/Height) of the image being created and optionally set its calibration and zoom (image magnification).

Channels to tabs

If multiple channels are present in the image, the created document will have each mono channel as a separate tab. If this option is not checked, a RGB image will be created.

Export time

If checked, this function exports Time grouping as a T dimension.

Export Z-grouping as Z-stack

If checked, this function exports Z grouping as a Z dimension.

Export Layers as Z-stack

If checked, this function exports Layers as a Z dimension.

Auto Minimum Height

Check this box to perform automatic min height detection during analysis. When auto min height is selected the software runs a short (100 periods in the middle of selection range) pre-analysis with relaxed identification constraints to determine the optimal min height for a given data set. This feature is not available when a Z Calibration data set is loaded.

Auto Fit ROI

Check this box to allow fitting algorithm consider variable number of pixels depending on width and axial ratio of each individual peak. When this check box is cleared a fixed size pixel rectangle (typically 5 x 5 pixels) is used for fitting. Auto Fit ROI is recommended for 3D analysis. Fixed size ROI is recommended for 2D analysis.

Minimum Height

The intensity of the smallest (dimmest) peaks (minus the local background of that peak) to be identified as molecules. Any object whose peak intensity is below this value will not be identified. Please see Determining Min and Max Height. If auto min height feature is selected the Minimum Height displays “auto” until the N-STORM analysis is performed at which point the “auto” is replaced with the actual calculated min height value.

Maximum Height

The intensity of the largest (brightest) peaks to be identified as molecules. Any object whose peak intensity is above this value will not be identified. Please see Determining Min and Max Height.

Baseline

Closed shutter (zero photons) pixel response. For Andor DU897 and for Hamamatsu Orca Flash 4.0 cameras it should always be 100. When currently loaded ND2 file was acquired with an officially supported camera, the CCD Baseline field becomes read-only and is automatically populated with the name of the camera manufacturer and the correct for the camera baseline value.

Minimum Width

The smallest possible width a spot of some intensity in the image to be identified as a molecule by STORM Analysis.

Maximum Width

The maximum possible width a spot of some intensity in the image to be identified as a molecule by STORM Analysis. A suggested default for 2D is 400nm. For 3D STORM, this should be slightly larger to take in account defocused molecules (above and below the focal plane). A suggested default for 3D is 700 nm.

Initial Fit Width

This value is used as a starting point for STORM Analysis for identifying molecules. For 2D STORM, this is the expected value of a diffraction limited spot. A suggested default for both 2D and 3D is 300 nm.

Max Axial Ratio

Fluorescent spots whose ratio of elongation in the X and Y direction is larger than this threshold will be rejected as a single molecule. It is typically set to 1.3 for 2D STORM and 2.5 for 3D STORM.

Max Displacement

This value sets the maximum distance (in pixels) that a molecule identified in one frame can be located from a molecule identified in the previous frame to be considered the same molecule and arising from the same Activation frame. The default value is 1. As mentioned in Z-Calibration section for Z Calibration files this value is ignored and 0 is used during STORM analysis.

Default

This button can be used to quickly bring Max Width and Max Axial Ratio in correspondence with current 2D/3D status of the data. For 2D Max Width is set to 400 nm and Max Axial Ratio to 1.3. For 3D these settings are 700 nm and 2.5 correspondingly.

3D

This checkbox indicates whether the dataset will be considered 2D or 3D during the analysis and subsequent display of results. This setting is typically applied prior to acquisition when the experiment is defined. During acquisition, it is stored to the ND file and read when the file is opened in the N-STORM module. However, this setting can be overwritten by checking or unchecking this box.

Fit Overlapping Peaks

This check box selects the fitting algorithm used for molecule localization. When cleared, regular single Gaussian peak fitting algorithm is used. When checked, the software attempts to discover partially overlapping peaks and perform Gaussian fitting that accounts for such overlap. This algorithm generally produces better results with fluorophores whose blinking efficiency is less than that of Alexa647 (for example Atto488) or in densely labeled and over activated areas where molecule peaks tend to overlap in their respective frames. Algorithm that accounts for overlap may take longer time to compute. A reminder is displayed at the beginning of analysis (excluding Test) when Fit Overlapping Peaks is enabled. This reminder can be disabled for the session.

ROI Statistics Table

The table summarizes the number of molecules in the image and in all ROIs. If multiple ROIs are selected, their features are shown per each ROI (separate column). More features such as the area, perimeter, center of mass and Niquist are calculated as well. To easily switch the units used in the statistics, click on the [px] <-> [µm] button in the top left corner of the table.

The first graph shows the Photon count chart which represents the peak intensity histogram in the ROI.

The second graph shows the Lateral localization chart representing the Width of the Gaussian peak.

The third graph shows the Ripley graph and cumulative histogram. Select one of the three graphs for molecule population - Ripley's K function (K), Ripley's H function (H), or CHD and set the Max search radius and Border correction. All the three graphs use the XY distance between molecules as the horizontal axis.

CHD (Cumulative Histogram of Distances)

For any given distance the value of the function is the number of molecule pairs in the ROI molecule population with distance equal or smaller to the given distance. Formal definition:

Figure 1495. 

where I_rI_r is the indicator function defined as

Figure 1496. 

nn is the number of points

rr is the max neighborhood search distance around any given point

d_ij is the distance between points i and j

Ripley’s K function

Ripley’s K function is similar to CHD except it is scaled by factor:

Figure 1497. 

Figure 1498. 

where λ is average density. In this form Ripley’s K function estimates the expected number of additional points within distance rr around a randomly picked point normalized per unit of density. λλ is the average density of the ROI population

λ = n/a

where aa is the ROI area.

Ripley’s K function is useful to explore cluster topology. When molecules form clusters, Ripley’s K function forms horizontal plateaus and steep rises (staircase look). For example, in the simplest case of two clusters, Ripley’s K function has one horizontal plateau.

Figure 1499. 

The horizontal axis of Ripley’s K function is the distance between molecules in a given pair. The vertical axis is proportional to the number of pairs in the population with distance not exceeding given. The first vertical rise on the left side comes from a number of close by neighbor molecules within each cluster. The position of the left edge of the horizontal plateau implies the size of clusters. As the distance increases further, the number of matching pairs does not increase until the distance reaches the distance between clusters. At that point the count begins to rise again, because larger distances between molecules of different clusters begin to contribute. Therefore the right edge of horizontal plateau suggests the inter cluster distance. Mouse cursor position in graph units is displayed in the bottom right corner as the mouse hovers over the graph area.

Ripley’s H function

Ripley’s H function can be defined in terms of Ripley’s K function.

Figure 1500. 

When points are randomly distributed, K function approximately follows the area of a circle function.

Figure 1501. 

The K function exceeds area function, when data points form clusters on a given scale. Conversely, the K function falls below the area function, when data points disperse (or form voids) on a given scale. The H function makes detection of clusters (or lack thereof) easier than the K function, because its value remains 0 under CSR (complete spatial randomness) condition. When the data points exhibit clustering, the H function becomes positive and forms a visible peak. When the data points are distributed uniformly, the H function becomes negative.

Figure 1502. 

All three cluster analysis functions quickly become computationally expensive as ROI population grows. Calculation time is greatly affected by other options such as maximum neighborhood search radius and edge effect correction method discussed below.

Max search radius edit box below the function selection buttons displays and adjusts the max neighborhood search radius. The button on its right side provides (and cycles through) three choices for distance units: nanometers (nm), micrometers (µm), and pixels. Pixels correspond to the original camera pixels (typically 160 nm). The largest supported search radius is 16 pixels or approximately 2.5 micrometers. The units apply to both the max search distance and the graph. When units change, the value of the Max search radius edit box automatically converts. The choice of units does not affect the length of calculation, but, when changed, commences the new calculation rather than converting results of the previous one. Reducing the Max search radius can greatly reduce the length of calculation.

Border correction is an edge effect correction attempting to compensate for the lack of neighboring points when the search is conducted near the ROI border. The circular search neighborhood then partially falls outside of the ROI. None simply skips the correction. This is the fastest method. Perimeter implements the popular correction method based on the fraction of circle perimeter within the ROI. Each measured distance carries a weight factor. When the circle around point i with radius equal to distance to point j is completely contained in the ROI the weight factor is 1. Otherwise the weight factor is equal to the fraction of the circle perimeter within ROI and is less than 1.

Figure 1503. 

During Ripley’s K function calculation contribution of each distance is divided by its weight factor, thus increasing when weight factor is less than 1 and therefore compensating for the possibly reduced neighbor count.

Figure 1504. 

Perimeter based edge effect correction is the most computationally expensive. Switching to a faster method, if acceptable, can greatly reduce calculation time.

Border correction method simply counts neighbor points outside of ROI that fall within max search radius or ROI points, but does not search their neighborhoods. All weight coefficients remain 1.

Figure 1505. 

Edge effect correction applies to CDH and H(r) functions in a manner similar to K(r) shown above.

When molecule data has multiple channels, cluster analysis can be performed across channels. In other words the distances are measured in one direction: from molecules in origin subset to molecules in target subset. These subsets can be defined as all molecules or molecules belonging to one of the channels. All combinations are possible. For example a two channel data set provides the following options:

Figure 1506. 

Start/End

These values specify the lower and upper Z limit of the calibrated dataset. Typically, these values will be smaller (by absolute value) than the top and bottom of the Z Stack used during the calibration process. These values are automatically updated during Auto Calibration.

Step

This value specifies the resolution of the calculated Wx/Wy calibration curve. Typically, data points that make the curve are spaced 1 nm (in Z direction) apart from each other.

Tolerance

The tolerance value specifies how far from the vs. curve a molecule can be and still be considered valid. Any molecules that are further from the curve than the specified tolerance distance will be placed into a special channel called ‘Z Rejected’. Z Rejected Molecules can optionally be displayed. The Default tolerance value is 1.5 nm.

Parabola Coefficients Values

There are two ways to determine the Wx and Wy calibration curves. The first method is for the software to capture a special Z series of a calibration slide (sub-resolution beads mounted to the cover glass). The elongation of every molecule is measured and the software automatically generates the curves.

The second method is to use the data collected from the Z-Series dataset and calculate the calibration outside of NIS-Elements using third party analysis software. The coefficients that were calculated outside of NIS-Elements can then be input into the parabola coefficients for Wx and Wy.

The defocusing curve formula is:

Figure 1508. 

is the lowest point in each graph or . and should be approximately the same. If they are not, one parabola is positioned higher than the other. It means points focus tighter in one direction than in the other likely due to spherical aberration.

is the focal depth of the microscope. Ideally it should also be the same between X and Y. Calibration fitting algorithm restricts it to stay below (and including) 5000.

is the relative to focal plane Z position where each curve focused the tightest. Ideally the values for X and Y should be the same by absolute value but with opposite signs (X negative and Y positive). This would mean that calibration is perfectly symmetrical around focal plane.

and values can be directly found in the and vs. graph as illustrated below.

Figure 1509. 

“A”, “B”, “C”, and “D” are higher order polynomial coefficients. A is the coefficient of the third order. B is the coefficient of the fourth order, and so on. Ideally all coefficients (A, B, C and D) should be 0. Typically coefficients A and B alone produce close enough approximation curve, so C and D are normally kept at 0 even in practice. There is an .ini file setting that removes the C and D constraint to deal with difficult cases. It's called “z calibration auto calib higher order corrections”. It is difficult to judge the quality of calibration by just looking at the values of these coefficients.

Auto Calibrate

This button automates the process of creating the Z calibration curves. It requires a Z-Calibration molecule list present (loaded or analyzed). Holding Ctrl while pressing Auto Calibration button causes the algorithm to ignore high density cluster localizations that may lead to peak overlap in out of focus frames. Ignored localizations are reassigned to Z Rejected channel that is otherwise not used with Z Calibration data.

Load/Save

Z Calibrations can be saved to a text file. These files can then be opened and applied to other 3D STORM datasets. These buttons are only available when ND2 data set is loaded. They work with a subset of calibrations pertaining to current data set. These calibrations may be different from the calibrations maintained by the system for newly acquired data sets. Calibrations loaded in this manner are called “foreign”. They become active (in effect for the subsequent 3D STORM analysis) and temporary override system or embedded nd2 file calibrations. They do not overwrite system calibration, which goes back in effect as soon as current nd2 file is closed. Foreign calibration can be permanently embedded into an nd2 file if at any point after foreign calibration is loaded the nd2 file is saved.

Graph

This button displays the menu with options for various graphs that visualize calibration curves and raw data and help evaluate results of current Z Calibration. There are eight types of Graphs available.

Figure 1510. 

1. Wx-Wy vs. Z – This graph shows the Wx and Wy calibration curves (cyan and yellow respectively) and the identified molecule’s Wx (red) and Wy (blue) values that pertain to currently selected in Z Calibration dialog channel tab. Generally the number of raw data points is limited to 3000. To display all points hold the Ctrl key while selecting the menu item.

2. Ax Ratio vs. Z -This graph will show the ratio of Wy/Wx on the Y axis and Z on the X axis. It also shows the identified molecule’s Wy/Wx ratio values (in blue). Generally the number of raw data points is limited to 3000. To display all points from the currently selected channel hold the Ctrl key while selecting the menu item.

3. Wx vs. Wy – shows the plot of Wx vs. Wy for the calibrated curves (in green) and of the individual localizations from current channel (in blue). Generally the number of raw data points is limited to 3000. To display all points hold the Ctrl key while selecting the menu item.

   Holding the Shift key while selecting this graph’s menu item displays the Z Rejected points in addition to the points from the currently selected channel tab. This helps checking how well the raw data follow the current calibration. Z Rejected molecules can be further categorized into those too far from the curve (yellow) and those with Z position beyond the calibration range (red).

4. Wx-Wy vs. Z (Frame Average) – this will show the same graph as above (#1) but only show the average Wx and Wy position per frame of the identified molecules. This graph is only available immediately after Z Calibration.

5. Ax vs. Z (Frame Average) - This will show the same graph as above (#2) but only display the average Ax (Wy/Wx ratio) per frame for the molecules. This graph is only available immediately after Z Calibration.

6. Wx vs. Wy (Frame Average) – This will show the same graph as above (#3) but only display the average Wx and average Wy per frame for the identified molecules. This graph is only available immediately after Z Calibration.

7. Axial Ratio vs. Period – This graph is useful for evaluating Z calibration data before performing Auto Calibration because it does not rely on any (previous or current) calibration. It simply displays the raw data points without any curve graphs. This graph is only available when Z calibration data is loaded and STORM analyzed. By default it displays up to 3000 points from the currently selected channel.

   To display all points from the current channel hold Ctrl while selecting the menu item. Sometimes the data contains outliers that affect scaling. To exclude outliers and see the data that more closely resembles the filtered set used for actual calibration, hold the Alt key while selecting this graph’s menu item. This option picks only 80% of points from the middle of the distribution in every frame and rejects outliers.

   To observe the effects of chromatic aberration on multichannel data hold the Shift key while selecting the menu item. This displays all channels at once.

   The channel colors used in this graph match the pseudo colors of the calibration data set.

8. Axial Ratio vs. Period (frame average) – This graph displays almost the same data as the one above (#7). The only difference it calculates the average for every frame and displays it as one point per frame. It has all the same options (Shift for multi channel, Ctrl for all points rather than 3000, Alt for skipping outliers). This is the most useful graph to evaluate raw Z calibration data before actual calibration.

   The software keeps track of multiple Z calibrations as they pertain to objectives with which they were acquired. During analysis the objective information is retrieved from the metadata and used to apply the correct calibration curves. If the calibration for data set objective is unavailable the software displays the warning and applies the default calibration. Similarly to Objective name software links calibration to reporter wavelengths and warns if calibration curve for any of the dataset wavelength is missing.

   The objective name pertaining to current Z Calibration is displayed in the title bar of the Z Calibration dialog.

   Calibrations for different wavelengths are affected by chromatic aberration. With a cylindrical lens in the optical path a given axial ratio of localizations happens at different Z positions for different wavelengths. Conversely a given Z position imaging with different wavelengths produces localizations with different axial ratios. When multiple wavelength calibrations are acquired and analyzed together, the software extracts axial chromatic shifts and maintains them along with calibration curves. When a newly acquired data set (acquired with the same objective and reporter wavelengths) is loaded and analyzed, the calibrated chromatic offsets are also applied to Z positions. The channel with the shortest reporter wavelength is used as a reference for chromatic aberration offsets.

   All graph dialogs have pop-up menu options to change the color of the background and grid lines. These colors are shared by all N-STORM analysis module graph dialogs and persist across NIS-Elements sessions.

   Similarly all graph dialogs share the ability to zoom by the mouse scroll wheel around the current cursor position and to pan the graph with a mouse drag operation.

   Graph image as well as numerical data can be transferred to a third party application via Windows Clipboard or exported into an image or text files on a disk. Both options appear at the bottom of the right click pop-up menu.

   All Z Calibration dialog buttons provide tooltip reminders.

ROI Source

This section defines the set of ROIs to be used. Automatically generated ROIs are arranged in a grid with definable dimensions (Grid). The Mix Manual and Auto generated ROIs button controls the mixing behavior. When the button is unchecked ()and manually defined ROIs exist, no auto generated ROIs are added. When the button is checked () or no manually defined ROIs exist, the grid of ROIs is generated according to grid dimensions. The left field is the number of columns and the right field is the number of rows.

ROI Filters

Not all generated ROIs are good for layer alignment. Only ROIs that pass through the ROI filter are considered.

First ROI filter is by the molecule count . ROIs with relatively small number of molecules are discarded. The Min Count threshold is defined as a percentage of the maximum single ROI molecule count among the ROI population. The button toggles the filter on and off. The filter is enabled by default.

The second filter is the Maximum average Nearest Neighbor distance filter (). If applied, it removes ROIs with average nearest neighbor distance greater than the specified percentage of the maximum average nearest neighbor distance if the molecules were evenly and randomly distributed throughout the ROI area without forming any structure or clusters. In practice this filter is not very useful because, with STORM, individual molecules get localized more than once. Even when fluorophores are evenly distributed, their localizations form tight clusters which significantly drives average nearest neighbor distance down, thus making ROIs that lack structure falsely identified as those containing a structure. This filter is available only in the Advanced mode and is disabled by default.

Autocorrelation density map

This group of controls provides several options that affect the autocorrelation algorithm input. Autocorrelation expects two similar signals. As it is used for layer alignment of localization data the signals are 2D density maps or histograms with bin values representing molecule counts falling within each spatial bin.

Z layer intersection ()or union ()choice affects the Z position filtering during histogram build. Intersection is chosen by default.

Another option ()is to merge ROIs for single autocorrelation and single offset result or () to perform separate autocorrelation for each ROI and then filter/average multiple results.

Outlier histogram bins can be optionally trimmed to avoid spurious autocorrelation offsets caused by occasional high density clusters. This option is enabled by default.

Figure 1511. 

Figure 1512. 

Autocorrelation offset filter

This filter is shown in the Advanced mode only. It is applicable to multi ROI alignment workflows that do not merge multiple ROIs (). This produces multiple offset results for a given pair of layers. The Offset Filter () checks how well multiple Cubes agree on the XY offset. DBSCAN algorithm is used to find a cluster that pertains to the subset of cubes that well agree with each other. Offsets obtained from other cubes are then ignored as outliers. The good offsets are averaged. To troubleshoot difficult layer alignment the Test Mode is available via the button. In the Test Mode intermediate data from each cube autocorrelation operation is available for close examination and alignment offsets are not applied to layers.