This procedure helps to ease the diagnosis by enhancing contrast through digital microscopes. Titrating your antibodies/dyes so that the stains are not extremely dark can be very helpful if you need to deal with overlapping stains.Īnother option is exporting a subsection of the image into Fiji to use ColorDeconvolution2 or other plugins to more carefully refine the vector values.Hematoxylin & Eosin (H&E) is a widely employed technique in pathology and histology to distinguish nuclei and cytoplasm in tissues by staining them in different colors. It could be 100% DAB, 100% hematoxylin, or something else. A black pixel allows no light, and therefor you have no idea what dyes were present. Even in a perfect world, with thin tissue slices, perfect staining procedures, and well separated stains (red, green, and hematoxylin), information is lost as the stains get darker. That means equal timing on the dips and washes per slide, and changing out the hematoxylin (and everything else) frequently enough. For stains like H&E, it is very important to keep the staining conditions constant. In this case I would need an Eosin only, and a Hematoxylin only stained sample - which I do not have. It is always best to generate your stain vectors from SINGLE stain samples. If you have access to red and green stains, those work much better with the hematoxylin as a counterstain since they will primarily - though not perfectly - affect information in different channels. The take home message here is ' be careful with what you think your analysis means.' Twice the DAB 'intensity' does not mean twice the protein.Īdditionally, because DAB shows up in the red green and blue channels, it is much harder to untangle from stains like hematoxylin, which are primarily blue. None of these reasons are particularly helpful from a quantitative analysis standpoint. DAB is commonly used both for historical reason (compare your staining vs other staining done with DAB), accessibility, and because it is usually dark and easy to see. One of the most difficult stains, and yet most common, is DAB ( DAB Wiki entry) ( DAB IHC description from abcam), which, in addition to being brown and thus crossing all three of the Red, Green and Blue channels for an RGB image, is a precipitate, which means it will not follow Lambert-Beer law and stoichiometrically absorb light that passes through it. DAB - ignore this section if you are lucky enough to not work with DAB You may be counting nuclei or cells via a stain like hematoxylin, or DAB nearby the nucleus via an expansion away from that nucleus, but the presence or absence of a stain, and in how many places is what most analyses will come down to. As such, the standard method of analyzing brightfield images is to attempt to isolate regions where your stain is present in sufficient quantities to meet some threshold, and then calculate the area of that stain.

That does not mean Fluorescence is perfectly quantitative - it is not for a variety of reasons - but brightfield is usually less so. We do the best we can, but generally brightfield analyses are much less quantitative than other types of imaging. Bulbs will age, darken, and their color warmth will shift. The color of the incoming light is unlikely to be perfectly uniform, even from an LED. The tissue itself will have some small absorbance, as will the mounting media. Color deconvoution gives us a way to ESTIMATE the amount of stain in a given pixel. Trying to analyze an image, though, we are interested in stains, not the original RGB values. As far as I know of, that does not exist, and the color of the lighting can impact the color of the stains we see on the screen, as can the color shading of the monitor. There are no pure color channels in brightfield, unless you manage to have a perfectly blue, red, or green dye. But these colors are usually not what we are interested in, unlike in fluorescence. Most brightfield images are three images in one, Red, Green and Blue all dropped on top of each other and presented to you on your screen via tiny Red Green and Blue lights. The stains themselves vary greatly in terms of their functionality. Why stain? And why DAB is not quantitativeīrightfield images, or at least the ones I will be discussing here, are originally fairly colorless pieces of tissue that have been stained with one or more dyes (I am not covering DIC/phase contrast or other unstained live cell analyses).

Imagej quantify he staining