Have a play and see which LUT helps illustrates the features in your image.
8 Colour Image processing
Images can have colour in three ways: a pseudocolour, as an RGB image or as a colour composite image.
Pseudocolour
A pseudo- coloured image is a single channel, (i.e. grey) image that has colour ascribed to it via a “look up table” or LUT (a.k.a. palette, colour table). This is literally a table of grey values (zero to 256 or 4095 whether 8-bit or 12-bit grey) with accompanying red, green and blue values. So instead of displaying a grey, the image displays a pixel with a defined amount of each colour. Differences in colour in the pseudo-coloured image reflect differences in intensity of the object rather than differences in colour of the specimen that has been imaged. For pseudocolour functions see later.
24-bit RGB images
The colours in RGB images (24-bit, 8-bits for each of the red, green and blue channels) and used to show multi-channel images. The colours are designed to reflect genuine colours, i.e. the green in an RGB image reflects green colour in the specimen, the differences in intensity of the green reflects differences in intensity of green in the specimen. There are several RGB functions in ImageJ. Native functions can be found in “Image/Color” and additional RGB functions can be found in “ Plugins/Colour functions ”. Another option would be to use Magenta rather than red in red-green-blue merge. An RGB merged image can be converted to “MGB” with the menu command ” Plugins/Colour Functions/RGB to MGB “.
Colour Composite Images
A colour composite can be thought of as similar to the way software like photoshop handle colour images – in ‘layers’, which ImageJ calls “channels”. The advantages with this type of image over RGB images are:
1. Each channel is kept separate from the others and can be turned on and off vial the ‘Channels’ dialog (Image>Hyperstacks>Channels or shift+z).
2. Each original channel can be kept as 16-bit.
3. More than 3 channels can be merged and kept separate. Each channel can be selected via the scroll bar at the bottom of the window.
4. The contrast and brightness of individual channels can be adjusted after merging.
Colour composite images can be converted to 24-bit RGB via the Channels dialog.
The disadvantage of the Composite image type is that this relatively recent ImageJ development is not supported by all existing plugins. Sometime your composite can revert to a multi-slice stack. To convert it back to a composite you can use the menu command “Image>Color>Make Composite“.
1 Merging multi-channel images
8.1. 1 Interleaved multi-channel experiments
Multi-channel experiments acquired on some systems are imported with the different channels interleaved, i.e. slice 1 is timepoint1-channel1, slice2 is timepoint1-channel2. The stack needs to be “De-interleaved” before it can be RGB-merged. This can be done with “ Plugins/Stacks-Shuffling/DeInterleave” and entering the number of channels in the dialog (typically “2”). The two stacks can then be merged via: “Image/Color/RGB merge”.
8.1.2 RGB colour merging
The native ImageJ function “Image>Color>RGB merge” can be used to merge red, green and/or blue channel images or Image Stacks
This reduces 16-bit images to 8-bits (based on the current Brightness and Contrast values) then generates a 24-bit RGB image.
An alternative to the normal Red-Green merge is to merge the images based on Cyan and Magenta, or Cyan-Yellow or any other colour combination.
This can aid visualisation of colocalisation due to our poor perception of red and green colours. The “ Plugins/Colour functions/Colour Merge XE “Cyan Magenta Merge : Tony Collins, Wayne Rasband ” ” function gives the user the option of using the ‘difference’ arithmetic processing on the image stacks you select. This is not strictly a merge (when cyan and magenta merge they produce white, not yellow) but facilitates visualisation of the separate channels (See Demandolx and Davoust, J. Microscopy, 1997 v185. p21). You can perform a true merge if you turn off the “Difference” option.
Run the plugin and select the two images to be merged. Select the desired colours from the drop-down options. uses the LUT that the image currently has (this is often the desired LUT). The “Difference” option performs a “difference” arithmetic operation rather than a “addition”. If the “Pre-sub 2 from 1” option is checked the second image is subtracted from the first prior to merging.
Merging trans mitted light and fluorescence images
Fluorescence and transmitted light brightfield images can be merged with the “ Plugins/Colour functions/RGB-Grey Merge ” XE ” RGB Grey Merge: Eugene Tkachenko ([email protected]) & Justin D. Pearlman” plugin. This plugin has been edited to include the option for users to pre-subtract a fraction of the fluorescence channel from the grey channel prior to merging. This can prevent a ‘washed out’ look to the fluorescence.
This also reduces 16-bit images to 8-bits (based on the current Brightness and Contrast values) then generates a 24-bit RGB image.
8.1.3. Merging images to a colour composite
The menu command ” Plugins>Colour>Composite merge ” opens a dialog to merge grey, red, green, and blue channels to a new composite image.
What’s the best red blending combination for Copic Marker beginners?
I asked three of my favorite marker instructors about which reds they recommend for first time colorers.
- Which reds offer you the best value?
- Which Copic reds are versatile enough to color many red items?
- Which reds do we teach with?
- Which red markers do we use in our own work?
Which Red Copics are Best for Beginners?
We highly recommend Copic’s R-Twenty red series for beginners and anyone building a small, versatile alcohol marker collection. R29, R27, R24, R22, and R20 are traditional reds which can be used for food, flowers, and holiday images. The lighter reds in this series can be used for pink objects.
Let’s talk to four Copic instructors to find out why.
Michelle Houghton’s Favorite Reds
The reds I most-often reach for are R22, R24, R29, R59. I also teach with them. This a very standard blending group but it’s one I have reliably used for a very long time.
However, I have shifted. Originally, I colored them from light to dark, now I tend to use them dark to light.
The R-Twenties are incredibly popular with colorers at all levels for a reason. R29 is a traditional fire-engine red, it’s not noticeably warm or cool, it’s just a beautifully balanced red.
Watch Michelle demonstrate her combination, offering tips for blending this combo both light to dark and dark to light.
In this next video, Michelle offers a more advanced combination, it’s beautiful!
Michelle is an elementary school teacher (BS Fine Arts, MA Education) with over 20 years of paper-crafting experience. She’s is a former Copic Certification Regional Instructor for Imagination International. Michelle runs the Copic in the Craftroom website and YouTube channel. Her Copic College events are favorites with Copic fans at all levels.. Visit her at Scrapweaver.com.
