Highlighting Color Correction

par | Oct 20, 2022

Often we want to visualize multiple structures within the same preparation, and if the colors are not registered, this can lead to erroneous conclusions. For example, if you are counting nuclei, the color correction does not likely matter, but when examining whether a particular protein is located within a synapse then it could. 

Multicolour imaging is a popular choice when trying to visualize multiple structures at once in a fluorescent sample. A simple example would be to stain a cell’s nucleus with blue DAPI and the cytoskeleton with a red rhodamine F-Actin stain. You may find it difficult to focus on both the rhodamine and the DAPI at the same time, with one always being blurry compared to the other. The reason for this might be that your objective is not corrected for imaging multiple colors, so you see the images focusing at different focal planes (along z).

Lenses that are specifically designed to focus multiple colors at once are described as Achromat, Fluorite, or Apochromat lenses. Achromat lenses are usually only corrected for single-color imaging and have no special description inscribed on the lens. Fluorite lenses are usually labeled “Fluor” and are corrected for 2-3 colors at once. Finally, Apochromat lenses are the most heavily corrected, indicated as “Apo” and can usually image 4-5 colors simultaneously.

Consider the objective lens shown in the figure below. This lens does not include any indication of correction, and only shows the magnification and numerical aperture. Because it is not color-corrected, red, green, and blue light are focused at different distances from the lens. This means that a multicolor object cannot be brought completely into focus.

Conversely, this next lens is labeled with the word “Apo”. This tells the user that the lens is corrected for at least four colors (Apo). As we can see, the lens focuses multiple colors to the same distance from the lens, meaning it can image a multicolored sample.

You may also find objectives labeled with the word “Plan” which indicates that the lens has a flat focal field across the whole field of view, useful for imaging thin flat sections. This is separate from the chromatic corrections so you may or may not see it on a given lens. In general, you will see one of the following cases written on a lens:

 

 

It may be tempting to reach for the highest corrected objective, but there are two major considerations that should give you pause. The first is the cost: lenses with higher correction have more internal optical elements and are much more difficult to make. This makes them very expensive. The second is the overall light transmission and sample brightness from a given lens. Since higher-corrected lenses have more elements (and thus more glass inside), they have more surfaces to absorb and reflect light. This can cause them to be less effective at transmitting light than a simpler design with fewer elements.

We hope you find this a useful guide when trying to determine the optimal lens for a given multicolor imaging application. Always contact the manufacturer to determine the exact colors a given lens is corrected for to be sure it matches your application!

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