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Biophys J, July 2002, p. 502-509, Vol. 83, No. 1

Laser-Scanning Coherent Anti-Stokes Raman Scattering Microscopy and Applications to Cell Biology

Ji-Xin Cheng,* Y. Kevin Jia,dagger Gengfeng Zheng,* and X. Sunney Xie*

 *Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, and  dagger SEG, Olympus America Inc., Melville, New York 11747-3157 USA

Laser-scanning coherent anti-Stokes Raman scattering (CARS) microscopy with fast data acquisition and high sensitivity has been developed for vibrational imaging of live cells. High three-dimensional (3D) resolution is achieved with two collinearly overlapped near infrared picosecond beams and a water objective with a high numerical aperture. Forward-detected CARS (F-CARS) and epi-detected CARS (E-CARS) images are recorded simultaneously. F-CARS is used for visualizing features comparable to or larger than the excitation wavelength, while E-CARS allows detection of smaller features with a high contrast. F-CARS and E-CARS images of live and unstained cells reveal details invisible in differential interference-contrast images. High-speed vibrational imaging of unstained cells undergoing mitosis and apoptosis has been carried out. For live NIH 3T3 cells in metaphase, 3D distribution of chromosomes is mapped at the frequency of the DNA backbone Raman band, while the vesicles surrounding the nucleus is imaged by E-CARS at the frequency of the C-H stretching Raman band. Apoptosis in NIH 3T3 cells is monitored using the CARS signal from aliphatic C-H stretching vibration.

Biophys J, July 2002, p. 502-509, Vol. 83, No. 1
© 2002 by the Biophysical Society   0006-3495/02/07/502/08  $2.00


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