Fluorescence microscopy image analysis
automation - time series - physiology

Measure intensity of whole view field with background masking

The pipeline calculates average fluorescence intensity for the whole view field after stabilization of the image time series, background subtraction and masking of background regions. The mask is calculated from maximum intensity projection of the time series therefore accounts for cellular movement during the timelapse. This pipeline is ideal for obtaining fluorescence intensities from low-light level single-channel recordings, where cellular movement occurs. Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

• Note: You have a two or more channels in your recording, but this pipeline processes only single channels. Alternatives:
  • Select channels one-by-one in the Multi-dimensional open dialog to process with this pipeline.
  • Use a two-channel pipeline instead.
  • Use the Pipeline window to create more complex pipelines starting with this pipeline.

• If the background is inhomogoneous the following protocols are suggested:
  • Measure intensities in single-cells using ROIs drawn by segmentation
  • Measure intensities corresponding to segments

• Select the 'Measure intensity of whole view field with background masking' pipeline.

Measure intensities in single-cells using ROIs drawn by segmentation

This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of a time lapse, creates ROIs from the segments and copies ROIs to the original image. Finally ROI means are plotted. Use: generic pre-processing for single-channel recordings, before single-cell analysis. Applicable to Ca2+-imaging (e.g. Fluo-3, GCaMP), reactive oxygen species imaging (e.g. DCFDA), GFP-based sensor imaging.

• Select the 'Measure intensities in single-cells using ROIs drawn by segmentation' pipeline.

• If the background is homogeneous, turn off image and local background subtraction.

• If the inhomogeneous background is due to illumination and a background image is available set Subtract reference image background = Yes, open a background image, and use the Context Menu to set "Set as Reference Image/Background / Dark Current". Such background image can be created if images form multiple x,y-positions of sparse cells using the
  Create background reference image for multiwell plate using median of wells pipeline.
• For generic removal of inhomogeneous background use Local background subtraction by median rolling ball = Yes.

Measure intensities corresponding to segments

Two pipelines are provided here to segment an image of cytosolic or nuclear stain into single cells or nuclei, and then measures intensities in the same or in an other channel, after performing background subtraction. .

• Select the 'Measure intensities using segmentation of the same image' pipeline.

• Select the 'Measure intensities using segmentation of another image' pipeline.

• If the background is homogeneous, turn off image and local background subtraction.

• If the inhomogeneous background is due to illumination and a background image is available set Subtract reference image background = Yes, open a background image, and use the Context Menu to set "Set as Reference Image/Background / Dark Current". Such background image can be created if images form multiple x,y-positions of sparse cells using the
  Create background reference image for multiwell plate using median of wells pipeline.
• For generic removal of inhomogeneous background use Local background subtraction by median rolling ball = Yes.

Measure fluorescence ratio of whole view field with background masking

This pipeline calculates average fluorescence ratio of all cells in the view field after stabilization of images, background subtraction and masking of background regions. The mask is calculated from maximum intensity projection images therefore accounts for cellular movement during the timelapse. The fluorescence ratio is calculated as the ratio of mean fluorescence intensities allowing working at low signal to noise ratio. This pipeline is ideal for obtaining fluorescence ratio from low-light level recordings, where cellular movement occurs. Applicable to Ca2+-imaging (e.g. Fura-2, yellow cameleons), other ratiometric ion probes imaging (e.g. PBFI, SPFI, SNARF-1, BCECF), reactive oxygen species imaging (e.g. HyPer, roGFP), and intramolecular FRET sensor imaging.

• This is a simplified pipeline. If the background is inhomogeneous the following protocols are suggested:
  • Measure fluorescence ratio in single-cells using segmentation-based ROIs

• Select the 'Measure fluorescence ratio of whole view field with background masking' pipeline.

• Enter the channel numbers for the numerator and denominator channels for the ratio calculation, e.g. Fura340 and Fura380, respectively.

Measure fluorescence ratio in single-cells using segmentation-based ROIs

This pipeline computes ratio image and shows it in Intensity Gated Pseudocolor, draws ROIs and plots ratio values as ratio of the average fluorescence intensities measured in the two source images for each individual cell. This pipeline stabilizes the time lapse (registers frames in time) and subtracts background. To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used. Then segments the maximum intensity projection of a time lapse, creates ROIs from the segments and copies ROIs to the original image. The fluorescence ratio is calculated as the ratio of mean fluorescence intensities allowing working at low signal to noise ratio. This pipeline is applicable to Ca2+-imaging (e.g. Fura-2, yellow cameleons), other ratiometric ion probes imaging (e.g. PBFI, SPFI, SNARF-1, BCECF), reactive oxygen species imaging (e.g. HyPer, roGFP), and intramolecular FRET sensor imaging.

• Select the 'Measure fluorescence ratio in single-cells using segmentation-based ROIs' pipeline.

• If the background is homogeneous, turn off image and local background subtraction.

• If the inhomogeneous background is due to illumination and a background image is available set Subtract reference image background = Yes, open a background image, and use the Context Menu to set "Set as Reference Image/Background / Dark Current". Such background image can be created if images form multiple x,y-positions of sparse cells using the
  Create background reference image for multiwell plate using median of wells pipeline, separately for each channel.
• For generic removal of inhomogeneous background use Local background subtraction by median rolling ball = Yes.

• Enter the channel numbers for the numerator and denominator channels for the ratio calculation, e.g. Fura340 and Fura380, respectively.

Measure intensities in single-cells in two channels with spectral unmixing

This pipeline corrects for misalignment between channels on the frame-by-frame basis, subtracts background, performs spectral unmixing with pre-defined coefficients, and stabilizes the time lapse (registers frames in time). To eliminate background the “Median of pixels below percentile of max projection” with the percentile given at “Background level” is used.
To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
Then ROIs are generated automatically using the nuclear stain as seeds, following the shape of the maximum intensity projection of the summed FLIPR+TMRM image. Therefore if a cell moves during the experiment, the ROI will contain the cell for the whole duration of the recording. The calibration is not affected by the amount of background recorded in the ROI.

• Select the 'Measure intensities in single-cells in two channels with spectral unmixing' pipeline.

• If the background is homogeneous, turn off local background subtraction.

• For generic removal of inhomogeneous background use Local background subtraction by median rolling ball = Yes.

Measure intensity of whole view field with background masking

• Open the recording and observe the cell density.

• Open an image series and observe the cell density.

• Set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant image and plot. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • The colored area in the resultant image is less than what is expected to be cells / appears to cover background

• If not using local background subtraction, set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant ROIs. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • Single cells are detected as multiple segments / Multiple cells are detected as single segments
  • Turn segment welding on / off in addition, to address the above.
  • Debris is detected as cells / Dimmer cells are missed
  • Only bright debris is detected / Brightest areas are clipped or fused
  • ROIs are smaller than cells / ROIs are too large or spilling over to background

Measure intensities corresponding to segments

• If not using local background subtraction, set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant ROIs. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • Single cells are detected as multiple segments / Multiple cells are detected as single segments
  • Turn segment welding on / off in addition, to address the above.
  • Debris is detected as cells / Dimmer cells are missed
  • Only bright debris is detected / Brightest areas are clipped or fused
  • ROIs are smaller than cells / ROIs are too large or spilling over to background

Measure fluorescence ratio of whole view field with background masking

• Open the recording and observe the cell density.

• Open an image series and observe the cell density.

• Set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant image and plot. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • The colored area in the resultant image is less than what is expected to be cells / appears to cover background

Measure fluorescence ratio in single-cells using segmentation-based ROIs

• If not using local background subtraction, set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant ROIs. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • Single cells are detected as multiple segments / Multiple cells are detected as single segments
  • Turn segment welding on / off in addition, to address the above.
  • Debris is detected as cells / Dimmer cells are missed
  • Only bright debris is detected / Brightest areas are clipped or fused
  • ROIs are smaller than cells / ROIs are too large or spilling over to background

Measure intensities in single-cells in two channels with spectral unmixing

• If not using local background subtraction, set frame-by-frame background subtraction as follows:
  • The cells are very sparse Use background level of 50 percentile.
  • The culture is subconfluent Use background level of 20 percentile.
  • The culture is confluent Use background level of 5 percentile.
• Run the pipeline, and observe the resultant ROIs. Adjust parameters if required and run pipeline again. Click the buttons below to adjust:
  • Single cells are detected as multiple segments / Multiple cells are detected as single segments
  • Turn segment welding on / off in addition, to address the above.
  • Debris is detected as cells / Dimmer cells are missed
  • Only bright debris is detected / Brightest areas are clipped or fused
  • ROIs are smaller than cells / ROIs are too large or spilling over to background