Having different digital images of various histological methods, especially immunohistochemistry, I want to quantify the results in terms of area, number of cells marked, and whatever else is possible.
Has anybody else encountered similar problems and found a good solution?
Hi. Maximilian. There are a variety of methods. For fluorescence IHC there are equipments that measure the intensity of the light and this is compared with the negative control of the same area ( consecutive histologic section), stained at the same time that the test slide. I have used long time ago and asume there are much better equipments to do that today. For IHC using other labelling methods there are software that can estimate the intensity, and allow specific counting of the labelled structures or cells within the region to be evaluated. In the past I have used "Neurolucida" for brain tissue, but there are other programs that can also be used. I suggest to explore the current options of software and consult with the manufacturers the feasibility of the application to your needs. Manufacturers often exhibit their products at Pathology conferences, you may explore if there is any of those conferences in your region.
In a simpler way it is also feasible to do semiquantitative assessments using measuring grids that are adjusted to ocular of any light microscope, e.g. cell damage/cell type, or structure immunolabled by specific biomarkers can be assessed by point sample intercept using a 40 objective and an eyepiece graticule for random sample analysis (Wang Biomedical KR-822BP 26 mm (www.wangbiomed.com)). The severity of the lesion can be assessed by grading from 0 to 4 the extent of the affected area using 20 objective.
Hope it helps, best Olga
Quantification of parameters such as area or number of cells marked is generally approached using sophisticated computerized image processing platforms.
I'm working on the same issue trying to quantify capillaries in different brain regions using ImageJ software I took several images from different brain regions see the below link for some answers.
https://www.researchgate.net/post/What_is_the_most_accurate_way_to_quantify_capillaries_in_a_mouse_brain_section
Best wishes
Sweilem
Hi. Maximilian. There are a variety of methods. For fluorescence IHC there are equipments that measure the intensity of the light and this is compared with the negative control of the same area ( consecutive histologic section), stained at the same time that the test slide. I have used long time ago and asume there are much better equipments to do that today. For IHC using other labelling methods there are software that can estimate the intensity, and allow specific counting of the labelled structures or cells within the region to be evaluated. In the past I have used "Neurolucida" for brain tissue, but there are other programs that can also be used. I suggest to explore the current options of software and consult with the manufacturers the feasibility of the application to your needs. Manufacturers often exhibit their products at Pathology conferences, you may explore if there is any of those conferences in your region.
In a simpler way it is also feasible to do semiquantitative assessments using measuring grids that are adjusted to ocular of any light microscope, e.g. cell damage/cell type, or structure immunolabled by specific biomarkers can be assessed by point sample intercept using a 40 objective and an eyepiece graticule for random sample analysis (Wang Biomedical KR-822BP 26 mm (www.wangbiomed.com)). The severity of the lesion can be assessed by grading from 0 to 4 the extent of the affected area using 20 objective.
Hope it helps, best Olga
Hi. Maximilian. here is another software program you can use: image analysis software, Northern Eclipse (Empix Imaging Inc., Mississauga, Ontario, Canada)
In attempting to use intensity of staining as a measure of the amount of antigen, you must show 1) that by some other measurement technique, the relationship between the amount of antigen and staining intensity, and VERY IMPORTANT demonstrate that your staining is linear with respect to the antibody molecules bound. The latter can only be shown by proper titration of the antibody. Use of too much antibody for staining will saturate the product (and this is true of both fluorescence and chromogen reactions). Here is a paper that shows another important feature: linearity of response is DIFFERENT when the antigen is present at high levels vs low levels. The most sophisticated imaging systems will NOT compensate for a lack of conducting an assay in the linear range.
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