Quick How To

1. Click Tools/Subtract Background, the toolbar icon or the Filters/Subtract Background function
2. Set Mean of pixels below percentile of max projection as Type, and 20-30 percentiles as Value
3. Checkmark images in the dialog or in the parameter bar and press OK or press the the button

More on this topic:

• Subtract background, normalize, OD

1. To draw ROIs use the following toolbar buttons: point ROI: line ROI: area ROI: magic wand ROI:
2. To select and move ROIs use the in the toolbar
3. To erase the selected ROI use DEL, the context menu of the Image Window or   in the toolbar.
4. Alternatively mark all cells automatically using the Plotting/Automatic ROI Drawing function or the Automatic ROI drawing with segmentation pipeline.
5. Optionally, to record data directly to Microsoft Excel, click Tools/Excel Data Window.
6. On the toolbar press to create a plot of fluorescence intensities. To configure plotting, select Plotting/Plot. Changes applied to this function will affect the operation of the toolbar button.
7. Save data by File/Save Excel Data, or by right-clicking the the Plot window.

More on this topic:

• Define ROIs
• Plot intensities, ratios of intensities
• Excel Data Window
• The following pipelines (in the Pipelines/Intensity Measurements menu) perform automatic pre-processing and intensity readout:
  • Measure fluorescence ratio of whole view field with background masking
  • Measure fluorescence ratio with automatic ROI drawing
  • Measure intensity of membranes
  • Measure intensity of whole view field with background masking
  • Measure of intensities in single-cells in two channels with spectral unmixing and using post-hoc seed image for segmentation
  • Measure intensities in single-cells using ROIs drawn by segmentation

1. Select the Editing/Align Series (Image Stabilizer) function in the Editing menu

2. Select the best signal to noise ratio channel as Image (A) by checkmarking in the left list in the center of the parameter bar

3. Select all images as Image (B) by checkmarking in the right list in the center of the parameter bar

4. The image registration is ready run now by pressing the button

More on this topic:

• Fixing imaging artifacts

• Several pipelines in the Pipelines/Intensity Measurements menu perform image stabilization, e.g:

  • Prepare two channel time lapse for intensity analysis and related pipelines
  • Measure intensities in single-cells using ROIs drawn by segmentation and related pipelines
  • Mitochondrial membrane potential measurement (TMRM/PMPI) and related pipelines

1. Mark any image as reference image by right-click/Set as Reference Image.  Alternatively use the I/O/Set Reference Image function
2. An image can by any of these three reference image types: Background / Dark Current, Blank for OD / Normalization, Histogram
3. Reference images are not closed by Close All, use File/Close All Reference Images to close them
4. Use reference images in the following functions:
   •  Tools/Subtract Background, and Filters/Subtract Background
   • Segmentation/Threshold
   • Segmentation/Advanced Segmentation
   • Appearance/Set scaling/LUT

1. Select a pipeline in the Pipelines menu, or open a pipeline file (*.ips) by drag-and-dropping on the main workplace, or by opening a new Pipeline Window in the PipelineDesigner menu
2. If you have open images, use any of these alternatives:
   • Press button in the main toolbar to run the pipeline in the top window. This is equivalent to the same button in the Pipeline Window.
   • Right-click the image and use Process this
   • Select a single image to as Image (A) in the the window press the toolbar button
3. If you have an open Multi-Dimensional Open dialog, use the button in the main toolbar, in the dialog or in the Pipeline Window to load recording and subsequently process with the pipeline
4. The button also serves automation, select Run Pipeline in All Stage Positions to evaluate the whole recording

 

Image Windows can contain only 2D time lapses. However analysis of XYZT stacks is supported by z-projection during reading the data set. Control the projection in the Settings tab of the dialog. Use Mean intensity projection, and projection of sub-stacks (Load specified frames only... and Use z-drift stabilizer checkboxes) if mesuring intensities in XYZT recordings.

1. Opening a single multi-dimensional data file (such as *.nd2, *.nd, *.lif, *_Sum.lsm) results a Multi-Dimensional Open dialog.
2. Select the actual well in the top of the Open tab and press Open to load images. Use the Close All Before checkbox and the Open Next button to walk through the recording
3. Optionally rename wells using the Plate tab, or individually using the pencil button.
4. Use the drop-down menu of the button to automate multiwell / multiple stage position data analysis
5. Optionally, walk through the data set using the button set to Clear and Run Next Stage Position, and using a visualization pipeline, such as Show three color overlay.

• If the data is arranged as one file per stage position, use multiple selection during opening or drag-and-dropping, and set the File merge box to Merge as Positions in the Open tab of the Multi-Dimensional Open dialog. 

1. The internal loader has limited format compatibility (TIFF, LSM, STK, ND2, INF files), but is substantially faster than using Bio-formats. Time stamps are correctly read. Not all features of the Multi-Dimensional Open dialog are available for each format.
2. The Bio-formats reader is compatible with a large array of biological formats, but is slower than the internal reader, All functions of the Multi-Dimensional Open dialog are supported. The time stamps may be wrongly read for certain formats.

To switch between Internal loader and Bio-formats:

• Use the File type filter of the Open dialog
• File/Set Folder Locations and Default Open Method menu point
• Note: Drag-and-drop open method follows the last Fie type filter or Default Open Method settings, but if an unrecognizable file is dropped on the workplace, it will automatically switch to Bio-formats.

1. Select the Appearance/Set Scaling/LUT function
2. Set Scale type to Fixed Value
3. Enter Min Value and Max Value
4. Either select images to scale as image (A)  and press the button
5. Or right-click each image and select the Process This menu point.

• Right-click the Image Window and select Copy Image or Copy Image with all ROIs
• Right-click the scale bar in the status bar of the Image Window and select Copy Scalebar to Clipboard
• Right-click the Image Window and select Copy Plot Image
• Use the File/Export... menu points or the I/O/Export function.

Right-click the Image Window and select Attach Overlay Image.

Are you processing a single frame or are you interested in numbers and morphologies of cells in each frame in a time lapse?

1. To perform segmentation only, use the Cells or Nuclei segmentation pipeline.
2. To segment and image and count cells, use the Whole well cell count and related pipelines.
3. Example pipelines for two step segmentation by first detecting nuclei and then cell boundaries (Pipeines/Intensity Measurements/Applications menu):
   • Plasma membrane potential and GFP intensity single-cell analysis using post-hoc seed image
   • Mitochondrial membrane potential measurement (TMRM/PMPI) with post-hoc ICC-based ROI drawing
4. To build up a step-by-step image analysis see details about segmentation here.

Are you processing a time lapse and do you want to follow segments (cells, nuclei) in time? Then you need to track segments.

1. To track moving cells or nuclei use the Track segments with one intensity channel pipeline.
2. To track segments in a time lapse, use the Segmentation/Track Objects function.

Use image segmentation to count cells. The following built-in pipelines segment images and transfer cell count data the Excel Data Window (Pipelines/Cell Counting and Colocalization menu).

1. To count cells in a view field use the Whole well cell count pipeline.
2. To count cells in a whole well by extrapolating cell numbers from a strip of tiled image across the well use the Whole well strip cell count pipeline.
3. To count cells in a whole well entirely imaged by tiled imaging use the Seahorse well cell count with nuclear stain pipeline.
4. To count cells in all wells of a microplate (multi stage position) recording, use any of the above pipelines, and the select the Run Pipeline on All Stage Position menu point in the pull down menu of the button in the main toolbar, the Multi-Dimensional Open dialog or the Pipeline Window.

To count cells in a segmented image and record the count in Excel, use one of the following functions:

• Plotting/Plot Morphological Parameters, Morphological parameter=number
• Special/Strip to Well Count

1. Create a plot as explained above at "How to measure and save fluorescence intensity values using ROIs".
2. Press the Range Markers in the status bar of the Plot Window.
3. Select the range or ranges in the graph by left-click where to calculate rates.
4. Right-click and select Calculate Rates in Marked Ranges.

To directly create rate plots:

1. Select the Plotting/Plot function.
2. In the Range markers for means or rates enter the range of frames where to calculate the rates, e.g. 1-10. Hint: ranges can be also copied to the clipboard in a range marked Plot Window by right-click Copy Range Markers.
3. Set Calculate rates at markers to Yes.
4. Press the or  main toolbar button to create a new plot.

The best way to calculate fluorescence ratios is dividing mean fluorescence intensities measured in ROIs. To perform this with a single command, use the Plotting/Plot Ratio function. Select the numerator and denominator as Image (A) and (B), respectively.

To calculate a ratio image, use the Math/Ratio function. Select the numerator and denominator as Image (A) and (B), respectively. Note that you can measure ROI means of ratios in the ratio image, but results will be a lot noisier than using the Plotting/Plot Ratio function.

The originally published high pass filter (Cell Calcium. 30.5:311-321) is applicable to 512x512 pixels images at 0.15-0.2 µm resolution:

1. To use this original filter, select the Filters/2D DFT Filter, and as Filter file locate \Image Analyst\Filters\the original X-rhod-1 mito filter.flt in the Documents folder.
2. Set Absolute to Yes.
3. If the recorded image is 512x512 pixels, set Preserve edges to Yes.
4. If the recorded image is smaller than 512x512, set Enlarge paper to Yes, and Preserve edges to No. To properly use the original filter, a non-quadrangular image has to be smaller than 512x512, and set the Enlarge image to power of 2 to 9.
5. Hint: use the Editing/Crop and Editing/Resample Image to match an image size and resolution to the original filter. Alternatively design a new filter for your recording below.

Creating and using new high pass (band pass) filters:

• Filtering is performed similarly to the above, but using the parametric Filters/2D DFT Butterworth BP Filter, where Cut On and Cut Off parameters are numerically entered based on optimization performed in the Tools/Set DFT Filter and Filter Optimizations dialog.
• To manually adjust a filter:

1. Open an image.
2. To test a filter, go to Tools/Set DFT Filter and Filter Optimizations dialog, Generate tab and select 2D DFT Butterworth BP Filter.
3. Set Absolute to Yes.
4. If the recorded image is quadrangular and sized as power of 2 (256x256, 512x512, 1024x1024) set Preserve edges to Yes.
5. If the recorded image is not quadrangular and sized as power of 2, set Enlarge paper to Yes, and Preserve edges to No. To properly use the original filter,  set the Enlarge image to power of 2 to a value so 2^this value is larger than the longest edge of the image.
6. Enter a higher cut off and a lower cut on value and press the preview button.
7. Adjust parameters and see the results.

• To numerically optimize a high pass (band pass) filter (will come with version 3.0.1):

1. Open an image.
2. To create a new filter with numerical optimization, go to Tools/Set DFT Filter and Filter Optimizations dialog, Optimization tab and select High Pass, Band Pass, Long Pass Filter. Follow this latter link for details how to perform optimization.
3. Copy the optimized Cut on and Cut off parameters to the Filters/2D DFT Butterworth BP Filter, Note that the optimized cut on and cut off frequencies (ω) are given in pixels, so set the Units of ω parameter to pixels.