A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images
Abstract
:1. Introduction
2. Results
2.1. Analysis of Photomicrographs from Each Brain Area
- A column with the remaining Skeleton IDs, without the duplicates (Figure 2, column G) [copy column B, paste it on column G, select new column Data tab > Delete duplicates].
- A column with the number of branches each Skeleton ID has (Figure 2, column H) [insert the function COUNTIF, in which the first condition comprises the values in column B, and the second one is the cell identification from column G, e.g., =COUNTIF($B$2:$B$5000:G2)].
- And a column for the sum of each Skeleton’s branches length (Figure 2, column I) [insert the function SUMIF, in which the first condition is the values in column B, the second is the cell identification from column G and the third condition is Column C, e.g., =SUMIF($B$2:$B$5000;G2;$C$2:$C$5000)].
2.2. Groups’ Analysis Per Area
- The number of branches per cell (Figure 4, column N), given by the [total branch number (column F) divided by the number of astrocytes (column E)];
- The number of cells per area (Figure 4, column P), calculated as the [number of astrocytes (column E) divided by the area (column D)];
- And the normalization of the total branch length per cell (Figure 4, column R), was obtained by the [normalization of the total branch length (column M) and by the astrocyte number (column E)].
2.3. Graphical Representation Data
- A mathematical model (Figure 5a) representing the most frequent number of the branches’ length, distributed by length intervals, and normalized by the cell number. An increased branch size comparatively to the control group is a potential marker of an inflammatory state, since branching and branch size are known to increase upon astrocyte activation [10];
- The number of astrocytes (Figure 5b), normalized per area. The fluctuation in the number of cells present in a tissue is a marker for tissue homeostasis changes;
- The mean branch length per cell (Figure 5c). The general increase in the branch length is another indicator of possible changes to tissue homeostasis;
- A correlation between the number of branches and their length that can be projected in a scatter-plot (Figure 5d), as a virtual cell size. This graphical representation provides a spatial perception of the increasing astrocyte size/activation, among treatments, visually providing more information about a possible change in the homeostasis of the tissue.
3. Discussion
4. Materials and Methods
4.1. Samples
4.2. Immunohistochemisstry
4.3. Image Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Conventional Analysis of Photomicrographs | Improved Semi-Automatic Method |
---|---|
Manual quantification | Macro-friendly, semi-automatic processing |
User subjectivity | Plugin definition precision removes user subjectivity |
Dependent on image quality and magnification | Applicable to low-magnification images |
Complex photomicrographs | Independent of photomicrographs complexity |
Inappropriate for large samplings | Appropriate for any sampling size |
Time-consuming (manual quantification of photomicrographs from one treatment group may take weeks) | Fast (photomicrograph quantification from one treatment group, and data trimming done under one week) |
Expensive hardware and /or software | Use of common hardware/software from a biology laboratory |
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Marques, S.I.; Carmo, H.; Carvalho, F.; Sá, S.I.; Silva, J.P. A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images. Int. J. Mol. Sci. 2023, 24, 4508. https://doi.org/10.3390/ijms24054508
Marques SI, Carmo H, Carvalho F, Sá SI, Silva JP. A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images. International Journal of Molecular Sciences. 2023; 24(5):4508. https://doi.org/10.3390/ijms24054508
Chicago/Turabian StyleMarques, Sandra I., Helena Carmo, Félix Carvalho, Susana I. Sá, and João Pedro Silva. 2023. "A Semi-Automatic Method for the Quantification of Astrocyte Number and Branching in Bulk Immunohistochemistry Images" International Journal of Molecular Sciences 24, no. 5: 4508. https://doi.org/10.3390/ijms24054508