The importance of MOLECULAR IMAGING of Lipid Membranes in Red Blood Cells (RBCs), isolated from whole blood, by Fluorescence Microscopy and Electron Microscopy

University of Ferrara e Laboratorio della Rete ad Alta Tecnologia (LTTA)
Directed by Prof. Luca Maria Neri, MD

PROTOCOLS AND RESULTS SHOWN IN THIS PRESENTATION ARE PROPERTY OF LTTA. ANY IMPROPER USE WILL BE LEGALLY PROSECUTED

For the BiophotOmics Project, the laboratory of Biomarkers, Biomolecular targets, and Personalized medicine in Oncology, headed by Prof. Luca Maria Neri, of the Department of Translational medicine, is performing the analysis of red blood cell membrane lipids both in normal and pathological conditions.
The assumption is that red blood cell membrane lipid content can be considered as a “molecular sensor” of the health status. In order to analyse the changing of the membrane lipid content, the help of molecular imaging becomes fundamental.
Erythrocytes, isolated from the whole blood, are stained by fluorescent probes on a single slide and then analysed by fluorescent microscopy. The probes used for this purpose are i) Cholera Toxin Subunit B (CT-B), which binds the ganglioside monosyalic acid (GM-1) and measures cholesterol levels, indirectly; ii) Filipin Complex, which, in turn, binds the free (not esterified) cholesterol in the membranes; iii) Laurdan, which takes place in the plasmatic membrane and is used to define the membrane fluidity status. In fact, thanks to the peculiar chemical structure, Laurdan shows a double fluorescent emission wavelength depending on the hydrophobicity of the micro environment, it is located.
The final images post-processed by the ImageJ software. Briefly, pixels of a region of interest (ROI) related to the plasmatic membrane, in 256 grey levels, are analysed to quantify the fluorescence from healthy and pathologic cells, which results different in the two conditions. These differences are related to the different lipid content in the cell membranes, where, usually, a pathologic condition results in a decrease of the membrane fluidity, allowing to describe new (bio)markers, for a not invasive diagnosis, of health status.

INTRODUCTION

FLUOROCHROMES

LAURDAN: its target is represented by glycerol bone of the acylic chains, forming the membrane phospholipids. The importance of this marker lies in its double emission, which depends upon the chemical environment. When Laurdan emits in the blue-green region in the visible spectrum, means that it is located in a more hydrophilic region of the membrane and, therefore, this area is more fluid. On the other hand, when Laurdan emits in the orange-red region, this means that it is located in a more hydrophobic area of the membrane, which is less fluid.

CHOLERA TOXIN SUBUNIT B (CT-B): the target is given by the Ganglioside-monosyalic acid (GM1), mostly located in the lipid rafts, within the plasmatic membrane and also in the outer layer. This is an indirect indicator of the esterified cholesterol content, whose lipid rafts are very rich. CT-B emits in the green region of the visible spectrum.

FILIPIN: the target is the free cholesterol in the membrane (non-esterified cholesterol). So that, the fluorescence intensity of filipin is a direct indicator of the cholesterol amount , within the plasmatic membrane. The more is the fluorescence intensity, the less the membrane will be fluid. Filipin emits in the blue region of the visible spectrum.

RED BLOOD CELL LIPID MEMBRANES FLUORESCENT STAINING PROTOCOLS

STAINING with LAURDAN, FILIPIN and CHOLERA TOXIN SUBUNIT B (CT-B) (1)

STAINING with LAURDAN, FILIPIN and CHOLERA TOXIN SUBUNIT B (CT-B) (2)

FLUORESCENCE MICROSCOPY

FROM MICROSCOPY TO IMAGE ANALYSIS

LAURDAN RESULTS

This image shows human RBCs, from healthy volounteers, stained with Laurdan as previously described. In the left part of the slide, the original staing. In the right part the post processing, performed with the ImageJ software. The color bar indicates the fluidity rate of the membrane. From -1 to 0 (red color), the membrane is not fluid, wherease from 0 to +1, the membrane is correctley fluid.

CT-B e FILIPIN RESULTS

This images show human RBCs, from healthy volounteers, stained with CT-B (left) and Filipin (right), following the protocol previously described. Then, the cells were imaged by fluorescence microscopy. The following post-process analysis aims at determining the difference in the fluorescence intenisty between healthy and pathology RBCs samples.

HEPATOCELLULAR CARCINOMA MOUSE MODEL

Hepatocellular carcinoma is a multistep pathologic process, characterized by the progressive evolution of hepatic diseases, causing cellular degeneration driven by fibrosis and cirrhosis, to the necrosis and tumor development.
In hepatocellular carcinoma mouse model, mice are treated with an intraperitoneal injection of Dietyl-nitrosammina (DEN) followed by be-weekly treatment with carbonate Tetrachloride (CCL4), in order to study all the different steps related to the tumor process.

CLINICAL RESEARCH

Clinical research is performed on human patients, in order to acquire more knowledges related to the diseases and to detect new treatments and to perform new device and new diagnostic procedures in order to improve heath care. According to that, voluntaries and patient affected to metabolic, neurologic and tumor diseases are going to be recruited allowing us to perform studies to confirm in vivo experiments.

CONCLUSIONS

REFERENCES

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  6. https://imagej.nih.gov/ij/