Hyperspectral Image Analysis of Live Cells in Various Cell Cycle Stages

In this study we have explored the use of hyperspectral imaging (HSI) to determine the cell-cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized by release from nocodazole or arrested in various cell-cycle phases with serum starvation (G₁), aphidicolin (S), or nocodazole (G₂/M). The live cells were then stained with the fluorescent DNA binding dyes Heochst 33342 or Dyecycle orange along with propidium iodide or Mitotracker green. Microscopic HSI data was then collected using the PARISS HSI system. Classified spectra were incorporated into spectral libraries; and all spectra acquired from each sample were correlated with library spectra to a user-determined confidence threshold, generating a unique spectral signature for each sample. Examination of these spectral signatures revealed that all cell cycle phases could be objectively differentiated. Ongoing studies employing other viable cell fluorescent dyes, and dyes in combination may provide more robust spectral signatures defining the status and condition of living cells.