Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

Area of Science:

• Biomedical Optics
• Chemical Imaging
• Cell Biology

Background:

• Confocal microscopy offers high resolution for biological imaging.
• Two-photon fluorescence microscopy enables deep tissue imaging.
• Raman microscopy provides label-free chemical information.

Purpose of the Study:

• To develop and demonstrate a hybrid confocal microscope integrating two-photon-excited fluorescence and Raman microscopy.
• To enable correlative imaging for enhanced biological sample analysis.
• To compare imaging resolutions of the combined system.

Main Methods:

• A confocal optical microscope was engineered to combine continuous-wave (cw) two-photon-excited fluorescence (TPEF) and spontaneous Raman microscopy.
• A 647-nm Kr-ion laser line was utilized for simultaneous excitation of Raman scattering and TPEF.
• The distribution of the UV-absorbing fluorophore Hoechst 33342 in apoptotic HeLa cells was investigated.
• Lateral and axial resolutions were assessed using Gaussian beam approximation and focal volume backprojection.

Main Results:

• The combined microscope allows fast fluorescence imaging to guide subsequent chemical analysis via Raman imaging.
• The distribution of Hoechst 33342 in apoptotic HeLa cells was successfully mapped using both modalities.
• Resolution characteristics of TPEF and Raman imaging modes were quantitatively compared.

Conclusions:

• The integrated system provides a powerful tool for correlative chemical and fluorescence imaging of biological specimens.
• This hybrid approach facilitates targeted chemical analysis based on fluorescence-guided region selection.
• The study validates the utility of combined TPEF and Raman microscopy for cellular studies.