Color separation of the light is known for already over 150 years. With practical application in color photography by of recording three monochrome images on glass panels using red, green and blue filters and later merged into color image on screen using the same color filters and white light source have started shortly after. In the modern days microscopy this simple technique can still find its use, saving significant amount of laboratory funding and efforts. By numerous reasons including better spatial resolution, higher light sensitivity and cheaper production majority of florescent microscopy systems are equipped with monochrome CCD camera. In many laboratories CCD based florescent microscope system is fully satisfy the majority of experimental needs, with only occasional necessity of color imaging typically for hematoxilin eosin staining and several other visible light based tissue and cell dyes detection. While high tier microscope systems usually have a both CCD and CMOS color camera installed, eliminating this problem the price difference between the models or the cost of purchasing additional visible light microscope or upgrading existing machine with additional CMOS sensor may be quite expensive.

Anton Lennikov dichroic_filters

The solution for this problem is to use RGB filters and separate the visible light into three color components. While commonly used in astrophotography this technique is less known to biomedical researchers. The price for RGB filter set on the consumer market may vary in between 20-200$ depending on the quality and specific properties, cheaper versions are usually quite good enough for the purpose. While use of specialized color filters is strongly advised it is even possible to use any transparent material of appropriate colors such as colored plastic as a improvised filter substitute, although resulting color composite may be of diminished quality with most notable affecting color balance as well as possible decrease in image sharpness.

Taking pictures

RGB_channels Anton Lennikov

First find the area on your slide you would like to record in color, fine tune focus and location. Then introduce red filter glass into optical system of the microscope (practical solution for filter placement might different depending on the microscope model and system design) record resulting monochrome image calling it %samplename%_r. Replace the filter to green and proceed with the acquisition of green and blue channels respectively as %samplename%_g and %samplename%_b. It is imperative that during this process sample will stay completely immobile without change in focus or stand location or color and visual artifacts will occur.

Resulting three monochrome files can be merged in Image J

study imageJ anton

Merge_channels Jpeg

(http://imagej.net/ImageJ) into full color image by opening all the files together and using command Image -> Color -> Merge channels.

Color Jpg

Human cornea H&E staining (EVOS FL microscope)

Resulting color image can be saved in any desirable format and further processed if necessary for brightness/contrast cropping e t.c.

Conclusion

Color separation with RGB filters and recording as monochrome channels is a viable technique for occasional color photography using CCD camera.