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How to make a scientific figure from microscopy data

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How to make a scientific figure from microscopy data
Scale bars (Pages 2-8)
Resizing and cropping (Page 9-12)
RGB splitting and merging (Pages 13-14)
Stacks and Montages (Pages 15-17)
Look up tables/pseudo-colours (Page 18)
Turning 16 bit data into 8 bit data/saving data in different formats (Page 19)
Scale Bars
Scale Bars
A general note about the scale of your images
The scale stays the same if you crop an image. So if 100Вµm = 103 pixels in A, 100Вµm = 103 pixels in B
The scale will change if you resize an image eg. From 1024 x 1024 to 512 x 512
So if 100Вµm = 103 pixels in A, 103pixels in B will now = 200Вµm, or 100Вµm = 51.5pixels
Scale Bars
In the tabs at the bottom of the image, select Overlay
Click on the ruler icon and draw a scale bar of desired length
You can change the colour and thickness of the line as desired
If you tick the Show all box on the right hand side of the Overlay tab, more options will appear
Scale Bars
Here you can change the size and style of the font, and adjust the position of the scale bar within the image
using the x and y coordinates
Click elsewhere in the image to hide the selection markers at the ends of the scale bar
To save the scale bar (or any overlays you have made), use the Save option in the Overlay tab. You can also
load previously saved scale bars by clicking the Load button. This way you do not need to re-create your
scale bar each time. NB. File type should be OVL
To export the image with the scale bar, choose File, Export, and Full resolution of the image window
Scale Bars
Alternatively, you can use the line tool to make a scale bar.
Select the Line tool and make sure the Measurement option is ticked
Draw a line of desired length, at an angle of 0 degrees
Un-tick the Measurement box to remove the text, then alter the colour or thickness of the line, and align in
the X and Y as before
Scale Bars
Image J
First set the scale of the image to microns instead of pixels. To do this go to Analyze, Set Scale
NB. We have already calculated the camera pixel to micron information for you. The excel file can be found
on the Z-drive. See below an example of the information collected from the timelapse microscope. Make
sure you select the correct microscope and objective.
Fill out the information in the Set Scale box, this will vary depending on the microscope and objective used
If you tick the Global option, it will set
the scale for all images currently open
Scale Bars
Once you have set the scale, you can then add a scale bar. Go to Analyse, Tools, Scale Bar
Decide the size of scale bar you want, as well as its colour, location, and whether or not you want text
NB. Image should be in RGB format
otherwise the scale bar may appear to be
the wrong colour.
To change the image format go to Image,
Type, and select RGB colour.
Once you are happy, remember to save your
image – File, Save As, Tiff
Scale Bars
Other image processing software
It is possible to draw scale bars on your images using other image processing software such as Photoshop,
Adobe Illustrator, or Inkscape etc.
The main principal is to draw a line of a known length and add this to all your images.
If your images were taken in Zen, draw a scale bar on just one of the images as described above, and export
the image. Then open this with your image processing software. Use the line tool to draw over the scale bar
in this image. You then have a line of correct length which you can add to all your images, provided they
were taken with the same objective and zoom.
Alternatively, use the information in our camera micron to pixel excel file, and work out how long to draw
your scale bar in pixels for it to be the desired length in microns. Then just draw a line that long.
Resizing and Cropping
Resizing and cropping your images
A general note about the scale of your images
The scale stays the same if you crop an image. So if 100Вµm = 103 pixels in A, 100Вµm = 103 pixels in B
The scale will change if you resize an image eg. From 1024 x 1024 to 512 x 512
So if 100Вµm = 103 pixels in A, 103pixels in B will now = 200Вµm, or 100Вµm = 51.5pixels
Resizing and Cropping
Image J
Details of the size of your image can be found in the
top left corner
This will be in pixels, unless you have set the scale of
your image, in which case it may be in microns, with
pixels in brackets
To resize your image, go to Image, Adjust, Size
Type in the size you want your image to be (in pixels)
NB. After resizing, the information in the top left of
the image will display the new image size
To crop your image, go to Image, Adjust, Canvas Size
Resizing and Cropping
Type in the size you want the cropped image to be
(in pixels)
You can change which part of the image you want to
retain by selecting from the drop down menu
If you select Centre, the image will be cropped on all
If you select Centre-Left, the image will be equally
cropped top and bottom, but only the right side of
the image will be cropped, retaining the left side of
the image.
If you select Top-Left, the image will be cropped
from the bottom and from the right.
And if you select Bottom-Centre, the image will be
cropped equally on the left and right, but only
cropped from the top…etc.
Original image
Centre Crop
Centre-Left Crop
Top-Left Crop
Bottom-Centre Crop
Resizing and Cropping
You can also crop by using the selection tools, and
highlighting the area you want to keep, then going to
Image, Crop
Selection tools
Merging and Splitting Channels
Merging and Splitting Channels
Image J
To merge red, green and blue colour images (RGB format), open the files you want to merge, then go to
Image, Colour, Merge Channels
In the Colour Merge window, select the
images that correspond with each channel.
Un-tick the box for Create Composite, and
tick the box Keep Source Images.
Remember to save the colour combine image
Merging and Splitting Channels
To split the channels of an RGB image, go to Image, Colour, Split Channels
Three grey scale images will be formed, one for each channel; Red, Green and Blue
You can then use the Lookup Tables to pseudo-colour your images
Stacks and Montages
Stacks and montages
Image J
To create a stack from a series of images, open the images you want to use, and then go to Image, Stacks,
Images to Stack. NB. Make sure you open the images in the order you want them stacked
Input a name for the stack, and type in a word or
abbreviation that appears in all the image names
Use the scroll bar at the bottom to move through the
Check that the images are in the correct order by
looking at the stack position and image name in the
top left of the image
Stacks and Montages
To turn a stack into separate images, go to Image, Stacks, Stack to images
Remember to save each of the separate images that
are created
To add or delete a slice go to Image, Stacks, Add or Delete Slice
Add Slice will insert a blank slice after the currently
displayed slice.
Delete Slice will delete the currently displayed slice.
To view a large stack or series of images which
exceeds the memory of the software, go to File,
Import, Tiff virtual stack
Stacks and Montages
To make a montage, open a stack containing the images you want in the montage. Then go to Image, Stacks,
Make Montage
Fill in the details about how you want the montage
to look
To make a montage using Metamorph, see the Making a Montage section of the Metamorph
guide; Page 4
Image J
To assign a pseudo-colour to your image use the LUT shortcut, or go to Image, Lookup Tables
Choose what colour to make your image.
Red, Green, Blue, Cyan, Magenta and Yellow are
standard colours.
The Fire lookup table displays pixel intensity by
colour, so higher intesity is yellow and red, and
lower intensity is blue. This is a good way to display
FRET images.
Image with Fire lookup table
To save the assigned colour go to Image, Type, and select RGB colour, then save as Tiff
Saving images in different formats
Saving images in different formats
Image J
To change the format of your images go to Image,
Type and select the format you want
NB. You can turn a larger file format, such as a 16-bit
image, into a smaller file format, such as an 8-bit
image, but you shouldn’t try to turn a smaller file
format into a larger one, as the software will have to
make up data information
You can then save the image in this new format
(File, Save As, choose file type)
Most of the time you will save as a Tiff, however
there are many other file types you can choose from
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