Simultaneous multichannel nonlinear imaging: combined two-photon excited fluorescence and second-harmonic generation...

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Nonlinear optical microscopy techniques like TPF and SHG offer unique contrast mechanisms.
• Imaging biological structures such as DNA and chromosomes requires high resolution and specific contrast.
• Combining different imaging modalities can provide richer information than single techniques.

Purpose of the Study:

• To demonstrate simultaneous two-photon excited fluorescence (TPF) and second-harmonic generation (SHG) imaging.
• To apply this composite nonlinear microscopy to image DNA and chromosomes.
• To show that combined TPF and SHG provide complementary information on biological material structure and nonlinear properties.

Main Methods:

• Utilized a single femtosecond laser source.
• Employed a scanning microscope setup.
• Implemented separate detection modes in reflection and transmission for simultaneous image acquisition.

Main Results:

• Achieved simultaneous TPF and SHG imaging of DNA and chromosomes.
• Demonstrated that TPF and SHG provide complementary structural and nonlinear property information.
• Obtained precisely registered pure TPF and SHG images due to simultaneous acquisition.

Conclusions:

• Simultaneous TPF and SHG imaging is a powerful composite nonlinear microscopy technique.
• This method offers enhanced insights into DNA and chromosome structure and nonlinear properties.
• The combination of TPF and SHG surpasses the information obtainable from either technique alone.