Introduction To Imaris Webinar February 2013 | Bitplane

Introduction to Imaris

February 2013

Questions

1a. Everything you do to that image data thereafter is only on the area of interest or is it applied to the whole file?

If you use resampling open, only that portion of the data set will be open, e.g. only a portion of the entire file will be opened.

1b. So then anything you do with it after, be it spot detection or surface, tracking, etc is only for that portion?

Correct, only that portion. IF you want to open the entire dataset, and interactively apply settings to a small portion, there is a "ROI" option in the Surfaces/Spots/Filaments/Cells creation wizard process which gives you the option to apply the settings to the entire dataset.

2. Why are you opening a new group?

For demonstration purposes at this point. However for general usage, you would create a group which allows you to export statistics from multiple objects into a single xls/xml file - or to look at the statistics of multiple objects at once.

3. Can you show how to extract each image with their respective time frame?

Kevin would need to show a time series, in which case there would be a time bar at the bottom -- the current sample image is only a single time point, so various time points cannot be extracted.

4. Is it possible to change that grey background to black?

Yes, Preferences --> Display --> Background Color (or Background Color 2).

5. With how many chanels can I work?

Imaris has no inherent limit to the number of channels, however most graphics cards can only display up to 16 channels at a time, but it is possible to have more channels in the file.

6. How do you open up the 'Display Adjustment' Toolbar? Or any toolbars for that matter. I'm new to Imaris.

Edit --> Show Display Adjustment, or CTRL+D on Windows, or CMD+D on OSX.

7. How do you know when it is appropriate to segment when detecting surfaces?

Segmentation is appropriate when you want to measure something. If you are trying to find out something the objects in your image - e.g. what is the intensity in your cell - what is it's xyz dimension. where it it's center of mass? then segmentation is appropriate

8. I am facing a problem with the Imaris that sometimes when I open an image it's not showing the image, just black volume window?

This is either a graphics cards problem, or a driver issue, please contact ussupport@bitplane.com for more assistance.

9. Do you plan to do further sessions like that?

Yes, but I'm not sure about the schedule at this time.

10a. If you have an image and want to detect thousands of spots within a stack i.e. 300nm in diameter - and it becomes overbearing for the program to do it all at once, would you recommend, say only processing 1/4 of an image at a time? If not, how would you recommend dealing with such a large data set?

Yes, that is a feature of the ROI function in the spots creation wizard. You can setup the parameters , and then "apply to data set finally" it is a check box in the creation wizard.

10b. The problem is when we do that, we cannot load the file in Imaris again and the program hangs

You change spots to 'center point' view, not using the 'sphere' view. Before you save it, it should re-open.

11. Do we always have to be consistent on applying a particular cut off to all the pictures analysed together?

That depends on a couple of factors - are you using the same settings during acquisition? how are you setting the threshold, are you using a fixed intensity value - e.g. absolute intensity threshold, do you have photo bleaching? In an ideal world, yes you would use the same thresholds, but often images have such different levels that we have to use a procedure to set the threshold which might vary between images. The important thing is to be consistent in the method you are using.

12. Is there database Mar?

Currently Imaris only supports Image Access for Image Databases, but stay tuned for exciting new developments comming soon!

13. Will deconvolution affect the quality of the automatic tracking?

In general, yes. Deconvolution improves object contrast, and this will often improve the signal to noise, which will improve object detection, which will thus improve tracking. Please note that if you use the wrong settings in deconvolution, you can introduce aberrations to your image which can impact tracking, so it is vital to undestand the entire imaging workflow. and settings.

14. How can we track volume over time?

If you have Imaris Track module, then you can track that. This is built into the Surfaces Creation Wizard.

15. If you have one channel detecting one object and a second channel detecting that object plus another object (i.e. imaging of neurons and astrocytes), can you adjust the settings such that you can deal with each object individually? Not sure if I worded this appropriately.

Depends on the exact configuration, with the Filament Tracer module, you can use One channel for dendrites and the second channel for Spines, if you are using the Cell object, it is specifically designed for multiple channel segmentation.

16. Is there a method for quickly applying the same surfaces setting to multiple images in a dataset and exporting to a CSV?

Yes, after you have created an object, there is an option in the 'Creation Tab' which allows you to "Remember Parameters" and then that can be saved as a XML file, and imported/exported.

17. Can we work simultaneously on multiple images?

It is generally possible to load up to about 4 copies of Imaris on a single computer, I generally do not recommend more than that. And you can use the same creation parameters, but they are not linked.

18. Where could we get more help or FAQs

The FAQ server - http://flash.bitplane.com/support/faqs/faqs.cfm is quite out of date, but better info is at the knowledge base: http://www.bitplane.com/go/knowledge-base/how-to or http://www.bitplane.com/go/support/resources

19. What is the max data load i.e. GB u can add to a batch?

In theory there should be no limit, but the default configuration you will need to adjust the Batch Memory configuration settings depending on the memory requirements for processing a single image. E.g. if it takes 4 gb of ram to process a single image, then you should adjust the Imaris Batch settings to use 4000 mb of ram from the default of 800 mb, and then you should be able to load any number of images, for Batch to Process.

20. When I export a confocal volume from the Leica software as .tiff, it also produces a text file which appears to contain all the image parameters. Can Imarus use that data file and import the parameters, such as the image scale?

Imaris directly supports the Leica LEI and Leica LIF file formats, so it would be better to use those formats directly, rather than exporting to .tif files

21. Is my username/password for the customer portal the same as for the webinar page?

No, the webinars use Adobe Connect, and those are seperate seperate user lists from the Bitplane Customer Portal. However all of the previous webinar recordings are available via the Bitplane Customer Portal, so if you want to see it again, please log in there, and take a look.

22. Is there a database that Imaris can tap into to automatically detect morphology etc.

Not currently. Imaris does not use genetic algorithms or neural networks to to segment the images, rather we use combinations of image processing plus threshold to identify objects. So those sorts of object properties would not be used by Imaris

23. Zeiss has a new format: czi (carl zeiss image). Will it be supported anytime soon?

The Zeis czi format is quite complex and has many variants. We can already open some variants of it and we will be able to open more variants of the format in the next versions.

24. How about .lsm?

We already support lsm files. If you have multiple series in one large lsm file you need to select the lsm files and click on the settings button. There you can then select which series you want to open.

25. It is currently not possible to plot statistics from one type of objects with statistics from another type of objects eg plotting the size of spots against filament length. When will that be possible?

At the moment it is only possible to add several Vantages to one combined Vantage Plot to combine different objects and adjust the scaling. In addition there is already a Feature request to allow as well combinations of different object types. Unfortunately we can not say when exactly this will be implemented. But I will add your comment to the corresponding feture request.

26. Can we adjust the opacity of the surfaces that we create?

Yes this is possible. You need to open the color tab. There is a slider where you can adjust the transparency

27. Does the ImarisXT allow automatic analysis of data?

For automatic analysis we have the Batch Coordinator. There you can e.g. set up surface detection parameters for one image and apply these parameter for multiple images in a batch.

28. Is there an easy way to extract data, e.g if you measure neuron length? Or is this only done manually?

In order to detect filaments you can use the FilamentTracer Module. There we have 2 algorithm that are able to trace filaments automatically. In addition there are as well semi-automatic and manual methods to trace the filaments. Once the filemantes are detected we provide many different statistics e.g. dendrite length that are calulated automatically.

29. Is it possible to handle datasets larger than the available RAM?

Yes it is possible to handle images that are larger than the RAM but it is not recommended. Since then the performance is slowed down due to the fact that we need to cache the data to the hard disk. In this case you should use an SSD for the temp files.

30. Does Imaris have a deconvolution alghoritm?

Imaris has a connection to AutoQuant which we distribute as well but there is no own deconvolution algorithm in Imaris

31. I have tracked 200 dendritic cells in a lung tissue slices. I would like to measure the distance of the tracked cells from the epithelial layer of the lung tissue. Can I do it with Imaris?

You could use the cell module and detect the epithelial layer as a cell and the cell as nuclei or vesicles. As a statistic value you can the have the distance to the epithelial/cell membrane.

32. Is that possible to resample two channels in different ways?

In Imaris both channels need to have the same size in terms of x,y, and z and t. So only in case you can resample the 2 channels to the same end size you can combine them later.

33. Is it required that before applying batch coordinator to all images within a data set should be captured on same setting, like laser intensity , zoom factor, stack width etc.?

It is not required but for sure it would be good in order to be able to use the same parameters settings. If the intensities are much variant the specified parameter will not work on all the images to be analysed.

34. What are the minimal hardware requirements for Imaris with all modules?

Our system requirements can be found at: http://www.bitplane.ch/go/support/system-requirements.

35. Is there a way to compare intensity statistics from a sample and a control analysis (from 2 different images)?

Unfortunately not for intensity. Other parameter that are not calculated on the volume data like area can be compared by combining the different scence files to a new combined scene and then e.g plotted in Vantage

36. Does the AutoQuant allows deconvolution for 3D data?

Yes AutoQuant works with 3D and 3D + time.

37. Sometimes the opened image is displayed 180 degree rotated. Where do I change orientation of the image?

Under the View menu you can set where you have the origin in the data.

38. Did you have new results from using a SSD as a cache drive and would two SSD can improve the seeking speed also like in the case of using normal Hard Drives?

We tested with one Solid State Disks (SSDs) OCZ Vertex 2 100 GB wich allowed about two times faster processing when used for storage of the Imaris TMP files than two Western Digital Raptors. Test procedure was to do a baseline subtraction of a 2hx2kx1k, 1 channel, 8 bit datasets on a Core 2 quad 3.0 GHz, 8 GB RAM system.

39. How about .lei files?

We already suppor the .lei files. You need to select hte lei file and choose the serie to open via the settings button.

40. Is there a 3D object counter? for example to count cells in an area?

Yes there is the option. Either you specify an ROI at the start of the wizard and detect the object only there. Then you have directly the amount of objects in the statistics

40. is there a 'home' button that resets the rotation to 0?

In the Rotate you can first use Apply instead of OK. THen you see a preview and only once you press ok the dats will be rotated. In addition you can use File - Revert to File or you use UNDO.

41. How do I reduce the speed when saving my movie.

The amount of frames in influencing the speed. With more frames and the rest set to default you will slow down the speed. You can then still use only some frames to specify the different keyframes

42. Does Imaris open tiled images?

You need to first stich the files before opening. There are some tools e.g. XUV Tools (Freeware) which support as well the imaris file format.

43. How can we quantify cytoplasm fluorescence of single cells?

We provide e.g. Intensity Sum, Mean, Max and Mean for each detected object. So you see these values for each object. In addition you can select the object of interest and choose Selection in the statistics tab.

44. Does Imaris open tiled images?

You need to first stich the files before opening. There are some tools e.g. XUV Tools (Freeware) which support as well the imaris file format.

45. How can we quantify cytoplasm fluorescence of single cells?

We provide e.g. Intensity Sum, Mean, Max and Mean for each detected object. So you see these values for each object. In addition you can select the object of interest and choose Selection in the statistics tab.

46. Is there a way to choose an area of interest that is not a square?

Yes you could add a surface, skip the wizard and draw a manual surface in the first and the last slide, generate the surface and then use mask and duplicate hte channels. Then you can use this new channels to detect you objects.

47. How do you label the channel with your own names, i.e blood vessel instead of red?

Just click on the name in the display adjustment. In the next window you can give a name to the channel.

48. What if the date set is hetrogenious like golgi stacks then how we put the diameter xy value.

The xy diameter is not rigourous. So if you choose e.g. a vlaue for the small object and use region growing you should be able to detect the different sizes.

49. In "spots" what is quality - what does is measure?

The 'Quality' is the intensity at the center of the spot in the channel the Spots was detected. You can find it in the Reference Maual in the section Spots - Creation Wizard - Classify Spots

50.In the spot can we change the sphereome other size like ellipsoidal etc.

Yes enable detect ellipsoids in Step 2 of the wizard in the spot detection.

51. I noticed several version of imaris on the computer of the presentor; would you need to have a subscription to obtain these? And if you dont have one, would that hamper performance of the last version?

The license is working downwards in a sense in case you have a license for 7.6.1 you can as well use e.g 7.5.3 or earlier versions. But we recommend to always upgrade to the latest version your license is valid for. But you keep older version installed in parallel. They do not disturb

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