In fluorescence microscopy, is it considered best practise to capture grayscale images and artificially colorize, or to capture color images directly?
Hi Patrick,
[Since fluorescence generally involves low levels of light, the sensitivity of the detector is important. The most sensitive detectors, such as sCMOS and CCD, tend to be monochrome. The filtering of the colour is then performed by a filter in front of the detector.]
As far as on-screen colour choice is concerned, it's really user-preference. I always advise people to use greyscale or green when looking at live images, since they are easier to see. (As you probably know, our eyes are most sensitive in the green region of the spectrum). Blue and red are not as easy to see, but there is no problem assigning the colour later for presentation / publication purposes.
To capture color images directly. It is useful to capture bright field and fluorescence images and then merge them. When you are staining for non ubiquitous marker the bright field will help you to identify where you marker is located within the cell. Never colorize artificially, save your files using RGB format. You can play with color intensity, saturation etc., after you've acquire your real images, with real fluorescence, but do it carefully. Hope this solve your question :)
It is just ok to acquire signals from 3 or more different color channels with using just one monochrome CCD camera, save them as tif images, then combine the signals from all the channels in the one image, and use false coloring for each channel signal.
Hi Patrick,
[Since fluorescence generally involves low levels of light, the sensitivity of the detector is important. The most sensitive detectors, such as sCMOS and CCD, tend to be monochrome. The filtering of the colour is then performed by a filter in front of the detector.]
As far as on-screen colour choice is concerned, it's really user-preference. I always advise people to use greyscale or green when looking at live images, since they are easier to see. (As you probably know, our eyes are most sensitive in the green region of the spectrum). Blue and red are not as easy to see, but there is no problem assigning the colour later for presentation / publication purposes.
Dear Patrick,
I agree with James, particularly if you have a low level signal that needs a sensitive camera, such as a cooled CCD, to capture.
Cheers!
Jill
Hi Patrick, finally it depends on your microscope equipment. If you have a cooled CCD or video camera you will see your fluorescence depending on the fluochromes that you have used in the real colour. Most of the highend microscopes like confocal or Apotome microscopes have high sensitive monchrome cameras and an additional computer program underlays the images with colours of your choice. Another importent point is the power of the light. if you are working with laser (confocal) you can play with the intensity to get good results evan if you have low signal intensity. If you are woking with fluorescence microscopes a 100 watt lamp is more effecient then a 50 watt lamp. Hope, this information will help you!
Hi :) When you capture the image, the camera detects only the light, not the color, as explained nicely above. When I capture images, I use an intensity heatmap (e. g. glow over/glow under) to adjust the laser intensity or the exposure time. In that way you get the right distribution of dim and strong signals. This is only displayed on the screen in false colors of a heat map, the pictures are usually taken in gray scale and you can add the color afterwards, as James described previously.
Best of luck! Tilo
One other related issue is the fact that the image on-screen can have its intensity changed independently of the acquisition settings (e.g. camera / laser etc.).
Although this is mostly helpful, it can cause problems / confusion if the user is not aware that this is happening - e.g. an image can appear saturated when it is actually very low intensity.
Most (all?) software should have some sort of "contrast" / "scaling" / "mapping" function to allow the user to adjust how the image is displayed on the screen.
Tilo's suggestion is a good one, but assumes this setting is set correctly - i.e. either the software or the user needs to know what value of signal is "too high" or "too low".
Capture grayscale images and artificially colorize, because high sensitivity cameras are not color cameras.
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