The pseudo-staining of Raman maps was performed by a semi-automatic approach. The clusters were grouped according to Raman features (peak at group-specific Raman shifts, see asterisks in Fig. ) mainly associated with five specific cellular components (cytoplasm, nucleus, carotenoids, MPO, HB) (Fig. ). Different color saturation levels within the same cellular component were assigned according to the peak intensities at the above-mentioned group-specific Raman shifts. We assigned lilac (light pink) to the cytoplasm and violet to the nucleus, aiming to resemble the appearance of stained samples used in clinics (Fig. ). We then considered that staining procedures are limited in detecting other specific molecules. For instance, MPO, an important hallmark to classify AML subtypes , is normally detected utilizing enzymatic-based or antibody-based labelling, on dedicated smears. Similarly, HB appears pink after staining but also other acidic proteins may have a similar appearance, thus preventing unequivocal detection. Carotenoids can be detected only using complex (e.g., isotope-based) labelling methods or by destructive (e.g., HPLC-based) approaches , and these are not available for routine diagnosis . For these reasons, the clusters associated with MPO, HB, and carotenoids were virtually colored in green, red, and orange, respectively, to increase contrast and legibility, and to eventually allow clinicians to easily recognize disease-specific biomarkers. A direct cell-to-cell comparison between virtual staining and real staining was not possible due to difficulties in staining and locating measured cells after Raman imaging. On the other hand, most of the general morphological features (e.g., cellular shape, nuclear shape, and nuclear-cytoplasmic ratio) emerging from virtually stained cells resemble those of stained cells (Fig. ) and are aligned with morphological features reported in the available diagnostic guidelines .