Two-Trace Two-Dimensional (2T2D) Correlation Spectroscopy of Lymphocytes in Infectious Mononucleosis

Raman spectroscopy has been widely demonstrated as a powerful tool for analyzing macromolecules characteristic of living organisms. This research focuses on a detailed spectroscopic analysis of lymphocytes affected by infectious mononucleosis caused by Epstein-Barr virus (EBV, HHV-4). As shown, at least 98% of the population is infected with EBV, making it one of the most common viruses. The main aim of this study is to evaluate the capability of Raman spectroscopy to distinguish between healthy lymphocytes from those infected with EBV at an early stage of infection. Blood samples were collected from patients with infectious mononucleosis who were receiving treatment at the University Hospital in Kraków. For comparative purposes, samples from healthy volunteers were also included. To guarantee detailed analysis, Raman spectra were acquired using two excitation wavelengths: 514.5 nm and 785 nm. The two-trace two-dimensional (2T2D) correlation method was performed to recognize the difference between EBV-infected and healthy cells. Initially, mean Raman spectra were calculated for healthy and infected cells and subsequently used to generate synchronous and asynchronous 2T2D correlation maps. Analysis of the asynchronous correlation spectra reveals distinct differences between healthy and diseased lymphocytes for both excitation wavelengths. For example, asynchronous peaks at 514.5 nm excitation indicate the beginning of immunoglobulin formation, while at 785 nm excitation indicate Tyrosine phosphorylation, which corresponds to activation of lymphocytes. The detection of specific biochemical alterations shows the potential of Raman spectroscopy as a non-invasive diagnostic tool in viral infections.