# Raman Spectral Signatures of Human Neutrophils: A Single‐Cell Approach

## TL;DR

This study shows how Raman microscopy can reliably detect molecular features of human neutrophils, offering a non-invasive way to study immune cells.

## Contribution

A standardized and reproducible Raman spectroscopy protocol for single-cell analysis of human neutrophils is established.

## Key findings

- Raman microscopy captured consistent spectral signatures of proteins, nucleic acids, carotenoids, and cytochrome c in neutrophils.
- Minimal interindividual variability was observed across 12 healthy donors using t-SNE and PCA.
- The method provides a robust framework for future studies on neutrophil metabolic states in health and disease.

## Abstract

With their noninvasive capabilities for molecular‐level analysis of biological tissues and cells, Raman spectroscopy and microscopy have become important tools in medicine and medical diagnostics.

In this work, we describe a reliable and repeatable technique for Raman spectroscopic profiling of human neutrophils in peripheral blood smears. 1200 single‐cell spectra from 12 healthy donors were obtained using Raman microscopy, and the results showed consistent spectral features of key biomolecules such as proteins, nucleic acids, carotenoids, and cytochrome c.

We used t‐distributed stochastic neighbor embedding and principal component analysis to assess interindividual variability; both techniques showed minimal spectral divergence between donors and high reproducibility. This study lays the methodological groundwork for upcoming Raman microscopy applications enabling non‐invasive assessment of immune cell states in health and disease.

Our results demonstrate the potential of Raman microscopy in clinical diagnostics, specifically for tracking the metabolic states of cells.

In this study, a standardized Raman microscopy protocol was established for single‐cell analysis of human neutrophils directly from routine hematological smears. Highly reproducible molecular fingerprints with low interindividual variability were obtained. This methodology provides a robust reference framework for future biophotonic studies of neutrophil states in health and disease.