Abstract
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.
Full Text
The Full Text of this article is available as a PDF (502.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bussolati O., Belletti S., Uggeri J., Gatti R., Orlandini G., Dall'Asta V., Gazzola G. C. Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells. Exp Cell Res. 1995 Oct;220(2):283–291. doi: 10.1006/excr.1995.1317. [DOI] [PubMed] [Google Scholar]
- Campagnola P. J., Wei M. D., Lewis A., Loew L. M. High-resolution nonlinear optical imaging of live cells by second harmonic generation. Biophys J. 1999 Dec;77(6):3341–3349. doi: 10.1016/S0006-3495(99)77165-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Campagnola Paul J., Millard Andrew C., Terasaki Mark, Hoppe Pamela E., Malone Christian J., Mohler William A. Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys J. 2002 Jan;82(1 Pt 1):493–508. doi: 10.1016/S0006-3495(02)75414-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng J. X., Book L. D., Xie X. S. Polarization coherent anti-Stokes Raman scattering microscopy. Opt Lett. 2001 Sep 1;26(17):1341–1343. doi: 10.1364/ol.26.001341. [DOI] [PubMed] [Google Scholar]
- Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
- Muller M, Squier J, De Lange CA, Brakenhoff GJ. CARS microscopy with folded BoxCARS phasematching. J Microsc. 2000 Feb;197(Pt 2):150–158. doi: 10.1046/j.1365-2818.2000.00648.x. [DOI] [PubMed] [Google Scholar]
- Müller M, Squier J, Wilson KR, Brakenhoff GJ. 3D microscopy of transparent objects using third-harmonic generation. J Microsc. 1998 Sep;191(3):266–274. doi: 10.1046/j.1365-2818.1998.00399.x. [DOI] [PubMed] [Google Scholar]
- Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]