Hyperspectral stimulated Raman scattering (SRS) microscopy
is a
robust imaging tool for the analysis of biological systems. Here,
we present a unique perspective, a label-free spatiotemporal map of
mitosis, by integrating hyperspectral SRS microscopy with advanced
chemometrics to assess the intrinsic biomolecular properties of an
essential process of mammalian life. The application of spectral phasor
analysis to multiwavelength SRS images in the high-wavenumber (HWN)
region of the Raman spectrum enabled the segmentation of subcellular
organelles based on innate SRS spectra. Traditional imaging of DNA
is primarily reliant on using fluorescent probes or stains which can
affect the biophysical properties of the cell. Here, we demonstrate
the label-free visualization of nuclear dynamics during mitosis coupled
with an evaluation of its spectral profile in a rapid and reproducible
manner. These results provide a snapshot of the cell division cycle
and chemical variability between intracellular compartments in single-cell
models, which is central to understanding the molecular foundations
of these fundamental biological processes. The evaluation of HWN images
by phasor analysis also facilitated the differentiation between cells
in separate phases of the cell cycle based solely on their nuclear
SRS spectral signal, which offers an interesting label-free approach
in combination with flow cytometry. Therefore, this study demonstrates
that SRS microscopy combined with spectral phasor analysis is a valuable
method for detailed optical fingerprinting at the subcellular level.