Correlated micro-spectroscopic labelling and analysis of leukocytes

We demonstrate identification of unstained leukocytes (white blood cells) in a full blood smear by discrete wavenumber mid-infrared (IR) imaging and spectroscopy. Using the novel technique of optical photothermal IR (O-PTIR) spectroscopy and imaging we achieve sub-micron spatial resolution and spectral resolution comparable to conventional IR-based techniques. Unstained full blood smears were prepared on glass substrates, and it was shown that leukocytes could be identified in high resolution O-PTIR images at two wavenumbers 1544 and 2922cm-1 acquired at a step size of 2μm. The discrete wavenumbers 1544, 1660 and 2922cm-1 correspond to the amide-II, amide-I and CH2 vibrational modes, respectively. After identification of leukocytes, an IR spectrum was obtained from each leukocyte and a second derivative spectrum was calculated to suppress broad spectral contributions of glass between 934 and 1228cm-1 which enable to resolve additional IR bands to identify chemical signatures. Single wavenumber images were acquired from leukocytes after visual inspection at 2922cm-1 followed by collecting five spectra within each leukocyte. Second derivatives of average spectra were calculated from both approaches and used for unsupervised hierarchical clustering analysis (HCA) of about ~160 leukocytes. Based on the number of spectra in each cluster, the actual leukocyte fractions were estimated in each sample. In summary, the technique of O-PTIR spectroscopy offers label-free, high resolution imaging with sub-micron spatial resolution of cells and spectroscopy to identify chemical signatures for the study of diseases and infections.