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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope
Published on: April 7, 2014
Comparison of different detection methods in quantitative microdensitometry
L Ermert1, A C Hocke, H R Duncker
1Department of Pathology, the Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany. leander.ermet@anatomie.med.uni-giessen.de
Insights
Quantitative immunohistochemistry requires reproducible methods. Vector Red substrate with absorbance microscopy offers excellent linearity and stability for reliable staining intensity evaluation in research and diagnostics.
Area of Science:
- Pathology
- Biotechnology
- Microscopy
Background:
- Quantitative evaluation of immunohistochemical staining is crucial for research and pathological diagnosis, such as Her-2/neu assessment.
- Reproducibility in immunostaining techniques and microscopic evaluation are essential for reliable quantitation of staining intensity.
Purpose of the Study:
- To compare different staining and microscopy techniques for quantitative evaluation.
- To describe and validate a Vector Red-based alkaline phosphatase immunohistochemistry staining procedure with microdensitometry for quantitative analysis.
Main Methods:
- Comparison of fluorescence microscopy, immunogold-silver epipolarization microscopy, and absorbance microdensitometry.
- Development of a Vector Red substrate staining procedure with alkaline phosphatase and custom absorbance filter microdensitometry.
- Characterization of staining intensity linearity using artificial standards with incorporated immunogold or alkaline phosphatase-conjugated antibodies.
Main Results:
- Vector Red demonstrated excellent qualities for quantitative microdensitometric evaluation, especially with absorbance microscopy.
- The Vector Red technique showed excellent segmentation, linearity over a wide range, and light stability.
- The method is applicable to paraffin-embedded tissues and cryosections, suitable for permanent mounting and long-term storage.
Conclusions:
- Alkaline phosphatase-based immunohistochemistry using Vector Red substrate and absorbance microdensitometry is a highly suitable technique for routine quantitative evaluation.
- This method provides reproducible and reliable quantitation of immunostaining intensity, enhancing its utility in research and diagnostics.
Abstract:
Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.
