# Applications of Raman Microscopy/Spectroscopy-Based Techniques to Plant Disease Diagnosis
## 5 Raman Microscopy in Single Cells--Single Microbial Cells  
Cells, whether individually or in groups, can be observed using various techniques. The most common method is light microscopy, which is frequently used in cell culture laboratories. It allows for quick assessment of the shape and coverage of cultured cells. However, this visual examination does not provide specific information about relevant molecules and detailed cellular structures. Other techniques, such as electron microscopy and fluorescence microscopy, are utilized to observe cells. Currently, confocal fluorescence microscopy is considered the most advanced technique for cell visualization. This method involves the use of fluorescent marker molecules that selectively bind to the structures of interest, enabling the specific visualization of different cell structures [47; 48; 49].  
The need for techniques that allow for chemically selective analysis to visualize and understand uptake is important in addition to visualization. Therefore, IR absorption spectroscopy and Raman spectroscopy are attractive methods for non-destructive and label-free imaging while maintaining chemical selectivity. Furthermore, both techniques can be combined with optical microscopy to enable powerful approaches to non-invasive chemical imaging. Due to the superior resolution of Raman microscopy, which makes it possible to analyze individual particles with dimensions of \(<\)1 \(\upmu\)m compared to IR microscopy (\(\sim\)10 \(\upmu\)m) and its applicability in aqueous environments, confocal Raman microscopy is now the predominant technique for cellular visualization [50; 51; 52].  
Therefore, the utilization of Raman microscopy in cell biology has definitively added to the visualization of cell biology for a series of biochemical species involved in cell division. Moreover, nowadays, this method contributes to cellular phenotyping. This is mainly because Raman spectroscopy can supply reliable information about the concentration of various amino acids and nucleotides, besides other components. Raman spectroscopy can highlight the concentration of both proteins and nuclear acids, separated from lipids and other cellular components [53].  
Raman spectroscopy can distinguish between different cell types and states (e.g., healthy vs. diseased cells) based on their unique spectral signatures. For example, Raman spectroscopy is used to detect cancerous cells and understand their biochemical changes, aiding in early diagnosis and treatment monitoring [54; 55].  
Raman spectroscopy is increasingly being used to study single microbial cells within populations, offering insights into their biochemical composition, metabolic states and interactions. This technique is particularly valuable for microbial ecology, biotechnology and medical microbiology [56; 57]. Raman spectroscopy can differentiate between microbial species and strains based on their unique spectral fingerprints. This is valuable for microbial taxonomy and diagnosing infections. This technique can analyze the biochemical composition of microbes at the single-cell level, providing insights into their formation, structure and biochemical composition. Therefore, Raman spectroscopy can be used to study microbial communities in their natural environments, such as soil or water, providing information on their metabolic activities and interactions [58; 59].  
Raman spectra can provide insight into the molecular composition of specific locations, allowing for the differentiation of components within a sample, such as organelles and cells in tissues. In addition to Raman peak mapping, advanced analysis methods like multiple curve resolution and independent component analysis (multivariate analysis techniques) can be used to visualize nuclei, lipid droplets and cell bodies individually. This offers valuable morphological information about samples without the need for labeling [46].