Imaris 9.7 Release Notes

Welcome to the Imaris 9.7 release notes. Please have a look to the overview of Imaris Release notes for information about prior released features and fixed bugs.

Version Date: February 9, 2021

Imaris 9.7 addresses two themes: Dynamics and Structure. New measurements and new plots provide useful information and analysis.

Imaris 9.7 facilitates the investigation of dynamics of individual cells by providing an easy mechanism to synchronize measurements from an entire population based on “events” independently defined for each cell. For example in a movie of dividing cells where divisions happen at different time points one can define an event for a cell when it undergoes cell division and then plot measurements synchronized by their time relative to the event.

Imaris 9.7 lets users address an important question: “Is there an interaction (spatial relationship) between spots and surfaces?” To answer this question Imaris computes the number of spots within a distance d of the surface as a function of the distance and compares the observed function with simulations of spots positioned under complete spatial randomness. Using this approach from spatial statistics users can study interactions between spots and surfaces.

Vantage Time Plots

For the study of dynamical phenomena we have added a Time plot to the Vantage module. It plots statistics values measured by surpass objects, i.e. spots, surfaces, cells, or filaments. The plot shows median values as well as 25 and 75 percent quantiles over time and it lets you easily set up a comparison between different surpass objects or between different classes in a single surpass object.

When objects are tracked Imaris automatically records “Time Since Track Start” statistics values. In the Vantage Time Plot you can choose this time as the x-axis to for example produce an MSD plot (mean squared displacement plot).

Other choices for the time axis come from the definition of events. For example when cells are tracked with the lineage tracking algorithm every cell division creates an event and this leads to time statistics called “Event Time Event = TrackSplit” which Imaris computes to enable plotting of statistics values around the time of cell division.

This plot shows the volume occupied by chromosomes before and after mitosis. To get a graph like this from many cells that divide at different time points it is essential to synchronize all measurements by their time relative to the event which is captured in the “Event Time Event=TrackSplit” statistics. Imaris 9.7 produces this measurement automatically.

Events

Imaris 9.7 introduces the concept of “events” to allow alignment of time series data by “events” on the time axis. Consider as an example three cells in the same image undergoing mitosis at different times, as shown in figure 1c. The graph in Figure 1a shows the intensity of the DAPI channel of the three cells plotted against acquisition time. Figure 1b shows the same graphs after aligning them by the events indicated on the time axis of Figure 1a.

Figure 1. Intensity of DAPI in three cells undergoing mitosis. a) Intensity plot versus acquisition time. b) Intensity plot against “EventTime” after defining events at the peak of chromosome condensation for each cell individually. c) Time series with the cell events indicated in pink.

Event Tab in the Creation Wizard

Events can be defined in the creation wizard of Spots or Surfaces. To do so one has to first classify objects into different classes and then one can set up “ClassChange” events for objects within a track when they “switch” from one class to another. This approach may seem somewhat indirect but it comes with the benefit of unlocking all the possibilities of the classification tools for the definition of events.

The event setup dialog lets you add any number of events and for each event you can define the class-change you are interested in. For example you may have set up an “inside” and an “outside” class based on the shortest distance between spots and surfaces to label spots as “inside” when their distance to the surface is negative and “outside” otherwise. You can then set up an event to capture when a spot changes from “outside” to “inside” and another event for when it changes from “inside” to “outside”. Each event you define produces a corresponding event time statistics for all those objects that are in a track which has such an event. This statistics value then becomes available as an x-axis for the Vantage Time plots.

TrackSplit and TrackMerge Events

When objects are tracked in such a way that tracks divide or merge --- through lineage tracking or connected components tracking or maximum overlapping cells or manual tracking --- then the objects immediately before a division or immediately after a merge will get a TrackSplit or TrackMerge Event and for all objects within a track that has a division or a merge an event time “Event Time Event= TrackSplit” will be computed.

Manual Event Editing

Events can be manually edited after object creation in the “Edit Events” tab. To assign an event to an object, select the object and press “assign” on the event you want to assign to that object.

Image Events

It is also possible in Imaris 9.7 to add “Image Events” to some time points of a time series image. These will also lead to event time statistics for Spots and Surfaces and Cells and Filaments so that again those event time statistics can be used as the time axis in Vantage Time.

To set up image events the time bar of Imaris has received an event toggle button that adds an event at the current time point if the current time point doesn’t already have an event. In case the current time point has an event the event toggle button removes the event from the current time point.

Existing image events are rendered on the time axis as yellow rectangles.

Event Time Statistics

When Events are defined for objects in a track Imaris 9.7 will automatically compute some new statistics for each object in the track.

TimeSinceEvent	Time since nearest previous Event. Before the first Event it has negative values.
TimeToEvent	Time to nearest next Event. After the last Event it has negative values.
EventTime	if(TimeSinceEvent < TimeToEvent) { EventTime = TimeSinceEvent; } else{ EventTime = -TimeToEvent; }

EventTime is computed for the purpose of plotting statistics “around the event”. It’s definition is somewhat arbitrary but it often fulfills the purpose of plotting statistics “around the event” quite well.

Surfaces Overlap Statistics

To help study the relation between two sets of surfaces Imaris 9.7 calculates statistics values measuring surface-surface overlap.

• Overlapped Volume to Surfaces Surfaces - The volume of the overlap region between this specific surface and all surfaces from the other Surface.
• Overlapped Volume Ratio to Surfaces - The ratio of the Overlapped Volume to Surfaces and the Volume of the specific surface.

Vantage Spatial Statistics

Imaris 9.7 provides some useful tools to study the question: “Is there an interaction between spots and surfaces?” and the follow up question: “At what distances do we observe attraction/repulsion?”

The starting point of this analysis is the measurement of the cumulative number of spots within a distance d from the surface and its less reliable1 counterpart the number of spots at a distance d from the surface.

Imaris 9.7 then simulates randomly positioned spots (the same number as observed) and generates a randomization envelope around the expected value of the simulations. This facilitates a useful comparison. Since the simulated spots are by construction neither attracted nor repelled from the surface they provide a useful null hypothesis and deviations from this null hypothesis indicate attraction or repulsion.

Inference from the cumulative graphs

Where the observed cumulative function exceeds the simulations it indicates “attraction” or more plainly that the observed number of spots within the respective distance to the surface is greater than what is expected from random simulations. Where the observed cumulative function is lower than the simulations it indicates “repulsion” or more plainly that the observed number of spots within the respective distance to the surface is lower than what is expected from random simulations.

Inference from the histogram graph

In the histogram graph we can observe at which distances from the surface spots occur in greater or lesser numbers than expected from random positioning. In the example it is at distances up to 3.5 micrometers from the surface that we observe a greater number of spots than expected from random positioning.

The probability density graph

The probability density graph is similar to the histogram graph in that it represents that density of spots at a distance from the surface. This graph is more smooth than the histogram because it is constructed using a kernel density estimate. The kernel smoothing is also applied to both the observed data and the simulated data (following [Duranton 2005]). The kernel width is displayed in the legend of the graph.

Randomization Envelope

The randomization envelope of the random spots is computed from 1000 simulations such that it encloses 98% of the simulations, 1% of the simulations were above the randomization envelope, and 1% below the randomization envelope. It is plausible to reject the hypothesis that observations are completely random when the observed curve lies outside of the randomization envelope.

Simulation Regions

Please take care to understand that the region for the simulation of random spots forms the basis for inference. It is deviations from complete spatial randomness in the simulation region that you can detect with the plots of Imaris9.7. Imaris9.7 provides 3 choices for the region within which random spots are simulated: Inside the surface, outside the surface or inside- and outside of the surface. Furthermore Imaris9.7 will automatically restrict the distance from the surface (outside and inside) within which spots are simulated to the distance that is displayed on the x-axis of each plot.

To visualize the region within which random spots are simulated you can use the Surface distance transform function (see below) to compute a distance transform from the surface and consider only distances in the range that is displayed on the x-axis.

The methods employed by Imaris 9.7 are based on well-established methods from the field of spatial statistics. In statistical terminology Imaris9.7 plots the “intensity” of the observed spots as a function of the shortest distance to the surface [Baddeley 2015 chapter 6.6]. In [Baddeley 2015 chapter 10] you can find a description of randomization envelopes and hypothesis tests. When building the methods for Imaris 9.7 the analysis of [Gomariz 2018] served as a model.

Image Intensity as a Function of Distance to Surface

Imaris 9.7 provides a plot showing the average image intensity of each image channel versus distance d from the surface. This plot lets you inspect whether the intensities in some channel change as a function of distance from the surface. Clearly we expect this for the channel from which the surface was computed. You may find that other channels also exhibit a change of intensity with distance.

Surfaces Distance Transform Native in Imaris

Via the “Mask Selection” or “Mask All” buttons on the Surfaces Edit dialog it is now possible to compute the Distance Transform. The result is a new floating point image with a channel that holds at each voxel the value for the shortest distance to the surface from the center of that voxel. The distance is computed inside and outside of the surfaces. The values of the Distance Transform inside the surfaces will be negative.

Unique New HostID Simplifies Workflow

The computation of an identifier for the computer on which a license is installed has been improved for Imaris9.7. Where previously users had to choose a HostId, Imaris now automatically computes a unique ID and no user choice is required.

Click here for instructions on how to update your license.

Acknowledgements

We want to thank Alvaro Gomariz and César Nombela-Arrieta for their support and for the discussions about spatial statistics and its use in Imaris.

Literature

• [Baddeley 2012] Baddeley, Adrian, Ya-Mei Chang, Yong Song, and Rolf Turner. "Nonparametric estimation of the dependence of a spatial point process on spatial covariates." Statistics and its interface 5, no. 2 (2012): 221-236.
• [Baddeley 2015] Baddeley, Adrian, Ege Rubak, and Rolf Turner. Spatial point patterns: methodology and applications with R. CRC press, 2015.
• [Gomariz 2018] Gomariz, Alvaro, Patrick M. Helbling, Stephan Isringhausen, Ute Suessbier, Anton Becker, Andreas Boss, Takashi Nagasawa et al. "Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy." Nature communications 9, no. 1 (2018): 1-15.
• [Duranton 2005] Duranton, Gilles, and Henry G. Overman. "Testing for localization using micro-geographic data." The Review of Economic Studies 72, no. 4 (2005): 1077-1106.
• [Ripley1981] Ripley, Brian D. Spatial statistics. Vol. 575. John Wiley & Sons, 2005.

Fixed Bugs

84 Bugs Fixed in Imaris 9.7
10010	Hard to determine which license is selected in Administrator - Replace QTableView with QTreeView in Imaris Administrator
10740	AddFilaments() in Imaris 9.1.x works like SetFilament() previously, i.e. deletes the current Filament
10757	OME Tiff files from Micromanager (1.4.23) does not get converted
10846	Edit Surfaces step of creation wizard is non-functional
10849	some .lsm files open with incorrect intensity values
11408	2D tiles of ND files are not correctly read in but superimposed
11472	Help for XTension Surfaces Split misleading
11659	Formula % of data set colocalized wrong
11774	Observed Folder Lists and Observed Folders can be lost when Imaris and ImarisViewer are opened at the same time
11794	XT Classify Spine is broken if there are more than two separate dendrite
11798	Statistics calculation happens too often while moving the frame
11801	Imaris crashes when saving this file
11851	Support Phase View Light Sheet Data Format
11916	Measurement Objects change mouse zoom behavior dramatically
11929	Measurement points are not placed exactly on surface of surface objects
11940	After creation of reference frame statistics are not displayed
11972	Imaris crashes when performing Rebuild Surfaces/Finish on PlantCell_with_objects.ims
12011	Manders' in Coloc Module has incorrect spelling
12012	Imaris Crashed When Trying to Change the Mapped Color of a New Channel Resulted from Channel Arithmetic of Ratio Two Channels
12021	Vantage full screen crash
12043	Imaris crashes when importing invalid ims file
12065	Tracks: Color Type > Statistics Coded > Advanced configuration doesn't have any effect
12085	Multi-image with no overlap results in incorrect Cell intensity statistics
12086	Imaris fails to convert these .tiff images
12114	Scalebar is showing wrong values if Snapshot size in 'Fixed' size mode is changed
12120	Imaris crashes on extract channel
12124	'Add Image' with customer data set changes the voxel size of the already loaded file
12137	OIF datasets with µ in name can not be opened
12161	Material Editor for Filament Points still contains "Diffusion/Specular/Emission" checkboxes
12174	Information about the Spots Quality filter is missing from the reference manual
12202	In Batch Setup, the mouse cursor does not change to indicate scalebar modification possibilites
12210	Help – About Imaris not immediately displayed
12227	ome tiff time interval is wrong
12232	Incorrect ome tiff file conversion
12235	Black stripes appear in the masked channel
12269	Channel visibility is not stored on "Save"
12271	Multi-image Surface ROI shows volume highlight in the wrong image
12278	Cannot hide channels in Batch Surface Creation in Slicer View
12296	Cannot close the preferences window
12304	Arrow keys do not function correctly when contour tracing
12318	No Context Help available when clicking in large empty space below Stitcher Preference License Dialog
12325	Object color in track editor didn't change with color tab
12326	Snapshot in track editor can't export a normal image
12330	Extract channel does not work on wasp image
12334	File Converter log file missing
12335	On MAC, installer buttons are white when using Dark Mode
12340	Need axis labels on the Filter 2D Classification step in creation wizard.
12343	'Camera' panel has no name when in tabbed view with Annotate
12354	Check mark 'Labels overriding base color' does not remember status
12365	XTensions do not start. Fatal error loading library ... mclbase.dll
12372	Crash when going back and forth between step Threshold and Filter Seed Points
12387	User-set data cache and number of threads are ignored
12390	Imaris 9.6.0 Programming Interface page title shows 9.7
12395	Imaris crashes when editing seed points and using volume preview
12396	Circle select doesn't work for 2D cells with nuclei
12399	Automatic reset of Display Adjustment position if off screen in all cases
12401	Surface - split using seed points broken - generating overlap
12405	Stitcher license dialogue reports 'Connection Failed!'
12406	select Coloc tab with 150% or higher windows scaling crashes Imaris
12418	overall statistics does not have Set and Class name
12424	Masking the Surface Export of ImarisCell fails with 2D image
12429	File open error with Scene for an IMS file - Could not load scene. bpTimes::FindTimeBegin(…) - IdTimeBegin 0 doesn’t exist.”
12430	Surface mask is not accurate in Imaris 9.6
12445	nd2 files fail to convert correctly
12447	Machine Learning Classification crashes Imaris in this image.
12452	Native Distance calculation crashes on this image
12455	Could not write File - Error when pressing 'Save' after drift correction
12458	Improve description of Memory and Data cache preferences
12460	Editing surfaces tracks crashes sometimes
12461	ImageJ TIFF second channel not read correctly - filled with zeros
12464	Anyone can use Vantage from "Plot" of Arena
12471	Missing MSVCR120.dll when installing Viewer 9.6
12481	No valid license dialog shows up 4 times
12502	Importing scene (with add components option) from same image creates correlation between duplicated components
12511	Imaris doesn't recognize .ims file converted from .tif since 9.6.0
12520	ICS2 file cannot be converted/loaded by Imaris 9.6
12543	9.6 reference manual, section2.9 is for old version vantage explanation
12545	Setting the Date/Time and Format in Windows to anything else other than English will cause issues in Wizards entering numbers with decimal point
12592	Topic id for Animation - Custom Rotation dialog is wrong
12611	Imaris freezes when showing Spots Average Statistics and changing time point
12634	Drift correction in 9.6 and 9.7 beta no longer works compared to 9.5.1
12644	"Channel extract” on 2D dataset crashes randomly
12653	XT ImarisApplication.SaveSnapShot() does not work
12673	'RIGHT MOUSE - Show Help' link broken for 'Preferences - Statistical Tests'