Quantitative image inspection and presentation

Prerequisites
Before starting this lesson, you should be familiar with:

Digital image basics

Lookup tables

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

Fluorescence microscopy data showing collagen secretion of tissue culture cells. Left: 0 hours secretion of collagen; right: 96 hours secretion of collagen.

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

Show activity for:

ImageJ Macro

/*
 * This ImageJ Macro 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
 * 
 */
 
run("Close All");

viewImage("https://github.com/NEUBIAS/training-resources/raw/master/image_data/image_inspection_and_presentation/xy_16bit__0h_dapi.ome.tif", "https://github.com/NEUBIAS/training-resources/raw/master/image_data/image_inspection_and_presentation/xy_16bit__0h_collagen.ome.tif", "0h", 1655, 958, 1928, 961);
viewImage("https://github.com/NEUBIAS/training-resources/raw/master/image_data/image_inspection_and_presentation/xy_16bit__96h_dapi.ome.tif", "https://github.com/NEUBIAS/training-resources/raw/master/image_data/image_inspection_and_presentation/xy_16bit__96h_collagen.ome.tif", "96h", 945, 1446, 1218, 1449);

// save all images as SVG for creating a figure in software such as PowerPoint or Inkscape
// CHANGE the outputFolder!!!
outputFolder = "/Users/tischer/Documents/training-resources/image_data/image_inspection_and_presentation/figure_creation_images";
setOption("ScaleConversions", true);
run("Export all images as SVG", "folder=["+outputFolder+"] exportchannelsseparately=Color interpolationrange=0.0 locksensitiverois=true");

function viewImage(dapiPath, collagenPath, treatment, px0, py0, px1, py1) { 
	// Open collagen image
	open(collagenPath);
	rename(treatment+"_collagen");
	
	// Adjust and display LUT
	setMinAndMax(0, 3500);
	run("Calibration Bar...", "location=[Upper Left] fill=White label=Black number=5 decimal=0 font=12 zoom=4 overlay");
	
	// Adjust and display spatial calibration
	// - in this case the pixel calibration is missing from the image metadata, thus we need to add it here
	cameraPixelSize = 6.45 // micrometer
	magnification = 20
	imagePixelSize = cameraPixelSize / magnification
	run("Properties...", "pixel_width=imagePixelSize pixel_height=imagePixelSize");
	Stack.setXUnit("micrometer");
	Stack.setYUnit("micrometer");
	run("Scale Bar...", "width=100 height=1 thickness=20 font=50 background=Black location=[Lower Left] overlay");

	// Open DAPI image	
	open(dapiPath);
	rename(treatment+"_dapi");
	setMinAndMax(0, 4000);  

	// Create merged image
	run("Merge Channels...", "c2="+treatment+"_collagen c3="+treatment+"_dapi create keep ignore");
	rename(treatment+"_composite");	// run("Calibration Bar...", "location=[Upper Left] fill=White label=Black number=5 decimal=0 font=12 zoom=4 overlay"); // ERROR
	run("Scale Bar...", "width=100 height=1 thickness=20 font=50 background=Black location=[Lower Left] overlay");

	// Create zoom in collagen image
	selectWindow(treatment+"_collagen");
	makeRectangle(921, 1278, 337, 337);
	run("Add Selection..."); // keep as overlay
	run("Duplicate...", treatment+"_collagen_zoom");
	run("Scale Bar...", "width=20 height=0 thickness=5 location=[Lower Left] overlay");
	run("Select All"); // to create an image boundary
	run("Add Selection..."); // keep as overlay
	

	// Create line profile in the middle of the zoom
	makeLine(20, getHeight()/2, getWidth()-20, getHeight()/2);
	run("Add Selection..."); // keep as overlay
	run("Plot Profile");
	Plot.setLimits(0.0,95.8,0,9000);
	
	
	// Close images that are not needed
	selectImage(treatment+"_dapi");
	close();
}

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 and max values for the treatment image

This can be done using the Set button of the B&C window. This will open another window Set Display Range. As an example, set Minimum displayed value and Maximum displayed value to 0 and 3500 respectively and select [x] Propagate to all other open images and press OK

If metadata is missing, add it manually to both images

Open Image > Properties...

Set Pixel width and Pixel height to 0.324 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 of B&C and set Minimum displayed value and Maximum displayed value to 0 and 4000 respectively and select [x] Propagate to all other open images and press OK

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 increase Thickness in pixels and Font 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 on 0h_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 reduce Thickness in pixels and Font 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 to 0 and YTo to whatever number and press OK

Select Rectangle tool from Fiji GUI, and select an ROI on 96h_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 selecting 96h_collagen image and doing Edit > 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 all SVG images

Export 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 a Blank presentation

Remove Text boxes or images, if present, by selecting them and pressing Delete key from keyboard

Drag and drop all the saved images from the folder into your Blank presentation

Select Intensity profile plots and ROI images, use right click menu and press Bring to Front

Resize and arrange all the images so that Intensity profile plots and ROI 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 pressing Group > Group

Use right click menu again on the Group image and press Save 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:

Learn more:

Forum discussion on collagen figure creation