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Spisák Krisztina

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Polgár Tamás Ferenc
Assessing the effect of photobleaching on morphometric parameters of different cell types in the central nervous system

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P51

Assessing the effect of photobleaching on morphometric parameters of different cell types in the central nervous system

Tamás F. Polgár1,2, Krisztina Spisák1,2, Zalán Kádár1, Nora Alodah3, László Siklós1 and Roland Patai1

1 Institute of Biophysics, Biological Research Centre, Szeged, Hungary

2 Theoretical Medicine Doctorate School, University of Szeged, Szeged, Hungary

3 College of Medicine, Alfaisal University, Kingdom of Saud Arabia

The sensitivity of immunofluorescence to the illumination time and intensity is well known; however, its effects on morphological parameters are less considered. Morphometric evaluation of different cell types could indicate the condition of the region of interest; for example, thickness and number of branches in microglia can imply their activation state.

Fluorescent detection using immunohistochemistry (IHC) targeting microglia/macrophages (fine structures) and neurons (bulk structures) was performed on neural tissues using different types of fluorophores while IHC with photostable diamonibenzidine was served as standard. Time series (0.5, 1, 2, 5, 10 and 15 minutes of illumination after taking the first image) were acquired from each spinal cord sections with the initial microscope settings. After standard mean intensity measurements, morphological parameters were extracted. Dynamic and relative total area microglial density were measured with a macro developed in our lab used routinely for quantifying glial activation. Fractal geometrical parameters were measured with the help of the Fiji plugin FracLac, and changes were examined between the initial image and the images containing faded structures.

Standard mean intensity measurements show different fading properties of different fluorophores. Dynamic and relative total area density comparisons show that in some cases all structures can fade to the point of becoming non-detectable after 2 minutes of illumination, while in some fractal geometrical parameters, more than 35 % differences can be observed.

Our results suggest that while fluorescent detection using IHC is an excellent method for localization and co-localization, for proper fine structure morphological measurements a photostable staining method is essential.

Acknowledgment

This work was partially supported by the Ministry for National Economy of Hungary through the GINOP-2.3.2-15-2016-00034 program. K.S. was supported from the UNKP-21-3-SZTE-73 New National Excellence Program of the Hungarian Ministry of Human Capacities. T.F.P. was supported by the EFOP 3.6.3-VEKOP-16-2017-00009 with the financial aid of the Ministry of Finance. R.P. was supported by the “National Talent Programme” with the financial aid of the Ministry of Human Capacities (NTP-NFTÖ-21-B-0203).