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Workshop Session - Basic Image Processing

Workshop Session - Basic Image Processing

LMIC - Image Processing Workshop, May 22, 2013: Building a Basic Figure

Preparation

Open PLOS to see example. We will be re-creating our version of fig 7D. Read figure legend to see how processing was documented.
Open Sample.tif to see my version of what you will create.
Open and read PLOS Author instructions. You should read author instruction for your journal BEFORE preparing figures. Also - Figures have to fit to print column sizes. Know what size you need before you start.
Open Photoshop, set Photoshop preferences to log your steps. Preferences/General/Check History Log/Choose Metadata, text file or both/EditLog Items = Detailed. Metadata is a file attached to your image. View from File/File Info/History.
Close all of this - you are ready to go.

Build a Figure

Duplicate original images
1. Open both cn01_08_prj-GFP and cn01_08_prj-Hoechst images.
2. Make copies of both images - Image/Duplicate.
3. Close original images.
4. Don't ever mess with your original images
Adjust brightness of cell nucleus
1. Click on Hoechst image - big cell nucleus is obscuring parasites we want to see
2. To turn down cell DNA (is this legal? Read figure legend in paper)
3. Select wand tool, set tolerance at about 66, click on Cell DNA, then shift click on bright spots until most of area is selected. To get remaining spots, click on Select/Modify/Expand (set expand to about 5).
4. Then Select/Modify/Feather (set to about 5) - to soften edge.
5. Go to Image/Adjust/Levels --> reduce output level to about 1/2 (255 --> 125), hit OK
6. Now Cell nucleus is at a similar level to parasite DNA.
Create Composite RGB Image
1. Click on GFP image, Click on Channels (tab next to layers), select all/copy
2. Click back on Hoechst image/Image/Mode/RGB color
3. Then click on Channels/Green/Paste
4. Then click on Red channel/Image/adjust/brightness-contrast (set both to far left to get rid of red color)
5. Click on RGB in Channels, then go back to layers. Now have a green/blue overlay image.
Get Crop tool, and crop to region of interest (around parasites).
Enlarge image for better view (command + on Mac)
Adjust color levels
1. Image/Adjust/Levels/pick color to adjust.
2. Green: Try moving input levels to match histogram.
3. Blue: very little adjustment.
Scale to 300dpi.
1. Image/Image size/Make sure resample image is NOT checked, change resolution to 300, hit OK
Make Composite
1. Duplicate Image --> Image/Duplicate
2. Image/Canvas size --> make width about 4X larger than image, position anchor where you want composite --> hit OK
3. Click back on single image we just made, get green channel - Channels/Green/select all/copy
4. Switch to 4X wide copy --> paste --> use move tool to place image.
5. Repeat for blue channel.
Make background white. Paint Bucket Tool/Select color = white/select background layer/click tool on empty region.
Crop to get rid of empty space.
Add scale bar
1. Set scale. Image/Analysis/Set Scale/Custom pixel length = 1
2. Logical length = 0.107 (from metadata), units = microns
3. Hit OK.
Add scale bar: Image/Analysis/Place scale Marker
1. This is kind of backwards. If you want a 3um scale bar you have to divide 3/.107 to get number of pixels (~28). Enter 28 in length, click desired text position and color and hit OK. You can use text tool to change number to 3.
Add figure labels and arrows.
1. Add labels if desired with the text tool, use move tool to place.
2. Add arrows with line tool. Set attributes at top, gear icon is where you get arrowheads.
Save
1. Save as Photoshop Image with Layers.
2. Save as Tiff for publication.
Check Log
1. If you checked text file - it is where you said it should be saved.
2. If you checked Metadata - View from File/File Info/History.
You are ready to publish!

Workshop Session - Basic Image Processing

LMIC - Image Processing Workshop, May 22, 2013: Building a Basic Figure

Preparation

Build a Figure

Light Microscopy Imaging Center resources