Raman-fluorescence hybrid microspectroscopy of cell nuclei

Raman-fluorescence hybrid confocal microspectroscopy of cell nuclei in different stages of nuclear development will be presented. Two-photon excited fluorescence microscopy enables fast detection of the morphology of the nucleus after staining with nucleotide sensitive dyes. Areas of interest are selected from the fluorescence image and subsequently analyzed with chemically selective Raman microspectroscopy. A single excitation source at 647.1 nm from a krypton-ion laser avoids spectral overlap between the Raman spectrum on the Stokes side and the two-photon excited fluorescence emission at the anti-Stokes side. Nucleotides are stained with DAPI or Hoechst 33342. The two-photon response of Hoechst 33342 is higher than that of DAPI. Raman microspectroscopy supports the finding that areas in live cell nuclei with low staining of Hoechst 33342 contain relatively high levels of RNA.