Quantitative image inspection and presentation
Prerequisites
Before starting this lesson, you should be familiar with:
Learning Objectives
After completing this lesson, learners should be able to:
Quantitatively inspect and present fluorescent microscopy images.
Motivation
For scientific discovery using microscopy it is critical to be able quantitatively inspect and present bioimaging data. This is important at many stages, ranging from looking at the data yourself, presenting the data to lab members and finally creating a figure for a publication.
Concept map
graph TD
ID("Image data") --> IS("Quantitative inspection")
IS --> P("Scientific presentation")
Figure
Activities
Inspect collagen image data
- This activity opens images where tissue culture cells are secreting collagen for different amounts of time. The aim is to display the images such that one can appreciate that after 96 h there is more collagen secreted than after 0 h. The aim is to make this display as quantitative as possible without doing an actual image analysis.
- Requirements:
- BioVoxxel Fiji Update site for saving images as SVG vector graphics
- NOTE (IMPORTANT): Please update to include BioVoxxel plugin (using
Help > Update > Manage update sites
) before proceeding to the activity
- NOTE (IMPORTANT): Please update to include BioVoxxel plugin (using
- BioVoxxel Fiji Update site for saving images as SVG vector graphics
Show activity for:
ImageJ Macro
ImageJ GUI
- Open collagen images 0h_collagen_secretion and 96h_collagen_secretion
- Adjust the display range of both images using
Image > Adjust > Brightness/Contrast...
- Try to select an intensity range for control image and then use the same
min
andmax
values for the treatment image
- This can be done using the
Set
button of theB&C
window. This will open another windowSet Display Range
. As an example, setMinimum displayed value
andMaximum displayed value
to0
and3500
respectively and select[x] Propagate to all other open images
and pressOK
- If metadata is missing, add it manually to both images
- Open
Image > Properties...
- Set
Pixel width
andPixel height
to0.324
micron
- Add calibration bar to both images using
Analyze > Tools > Calibration Bar...
Location
-Upper Left
Fill color
-White
Label color
-Black
Font size
-12
Zoom factor
-4
[x] Overlay
- Open dapi images 0h_dapi and 96h_dapi
- Adjust the display range of both images using
Image > Adjust > Brightness/Contrast...
- Use
Set
button ofB&C
and setMinimum displayed value
andMaximum displayed value
to0
and4000
respectively and select[x] Propagate to all other open images
and pressOK
- Merge channels belonging to the same image (e.g. “*_0h_collagen.ome.tif” belongs to “*_0h_dapi.ome.tif”) using
Image > Color > Merge Channels...
. and in the settings:
- Set collagen channel as
C2(green)
- Set dapi channel as
C3(blue)
[x] Create composite
[x] Keep source images
- Add scale bar to each image using
Analyze > Tools > Scale Bar...
and increaseThickness in pixels
andFont size
to suit your needs
Thickness in pixels
-20
Width in um
-100
Font size
-50
Location
-Lower left
[x] Overlay
- Close
dapi
single channel image windows- Select
Rectangle
tool from Fiji GUI, and select an ROI on0h_collagen
image (grayscale)
- Add it as overlay on the image using
Image > Overlay > Add Selection...
- Duplicate the selection to create an ROI image using
Image > Duplicate...
[] Ignore selection
- Note: The overlay box stays on the original image even if you click elsewhere
- Add scale bar to ROI image using
Analyze > Tools > Scale Bar...
and reduceThickness in pixels
andFont size
to suit your needs- Select a
Line
tool from Fiji GUI, and draw a line in the middle of ROI image- Add it as an overlay on the image using
Image > Overlay > Add Selection...
- Plot intensity profile along this line using
Analyze > Plot Profile
- Set the y-axis range using
Set>> Set Range...
- Set
YFrom
to0
andYTo
towhatever number
and pressOK
- Select
Rectangle
tool from Fiji GUI, and select an ROI on96h_collagen
image (grayscale) and repeat the procedure above
- Note: You can use the same ROI as above by selecting the ROI rectangular box on
0h_collagen
image and then selecting96h_collagen
image and doingEdit > Selection > Restore Selection
- To save all the images in the high quality, go to
Plugins > BioVoxxel Figure Toolbox > Export all images as SVG
Target folder
- folder of your choice to save allSVG
imagesExport channels
-Color
Lock critical ROIs [x]
Create collagen data figure
- Assemble the individual images that were prepared for quantitative data presentation into a figure
Show activity for:
Powerpoint
- Run
PowerPoint
and open aBlank presentation
- Remove
Text boxes
or images, if present, by selecting them and pressingDelete
key from keyboard- Drag and drop all the saved images from the folder into your
Blank presentation
- Select
Intensity profile plots
andROI images
, use right click menu and pressBring to Front
- Resize and arrange all the images so that
Intensity profile plots
andROI images
belong to their respective images.- When all images are arranged correctly, press
Ctrl + A
to select all images and group them using right click menu and pressingGroup > Group
- Use right click menu again on the
Group
image and pressSave as Picture
- Select a file name and folder and save as type
SVG
Assessment
Fill in the blanks
- TODO ___ .
- TODO ___ .
Solution
- TODO
- TODO
Explanations
Follow-up material
Recommended follow-up modules:
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