AML M2 cells studied by Raman imaging show a further decrease in nuclear-cytoplasmic ratio (Fig. ) and the presence of strong MPO Raman features occupying the entire cytoplasm, most of the time overlapping with the nuclear region (Fig. ). These signs are also consistent with diagnostic evidence related to AML M2, reporting the increase of mature cells, now called promyelocytes, and reporting MPO granules over the entire perinuclear region. AML M3 Raman cells show very intense and largely diffuse MPO signals with morphological features like those observed in M2 but presenting a different cluster distribution. It is important to note that in M1, M2, and M3, when MPO (green) clusters colocalize also with the nuclear regions in the center of the cell, DNA signals are still visible at the spectral level (788 cm -1 ). This agrees with the presence of the nucleus in such type of cells, but since MPO granules are very abundant and their Raman signals are very strong due to pre-resonance effects 20,47 the clustering analysis assigns most of the pixels to MPOcontaining clusters. Moreover, as seen in (Fig. ), MPO clusters have a different distribution over M1, M2, and M3, with the exclusive presence of cluster #14 in M3. In detail, cluster #14 (Fig. , dark green) shares some features with cluster #10-13, including a very strong peak around 1578 cm -1 , associated with MPO. At the same time, cluster #14 is characterized by relatively high intensity of Raman peaks around 681, 741, 992, and 1028 cm -1 , if compared with cluster #10-13. Further studies and an enlarged population sampling will be needed to investigate the peculiarity of these spectral features in M3 cells. Guidelines associate M3 with the presence of abnormal and hypergranulated promyelocytes, described as irregular and often kidney-shaped cells, with strong MPO-staining reaction and with MPO granules that may totally obscure the nuclear cytoplasmic margins 2 , in agreement with our data. In summary, the increasing maturation of cells, mostly represented by an increasing amount of MPO signatures and a decreasing nuclear-cytoplasmic ratio, is clearly visible when passing from AML M0 to AML M3.