Confocal Fluorescence Microscopy
Abstract
This article describes basic approaches to image materials by means of optical microscopy with fluorescence markers. The fluorescence microscopy is used to image materials in which the contrast cannot be achieved by light absorption, scattering, or birefringence. The fluorescence microscopy benefits greatly from a confocal mode of observation that eliminates signal coming from outside the small region of sample that is probed by a tightly focused laser beam. By scanning the focused laser beamthrough the sample, one obtains a 3D map of density of fluorescing molecules staining the sample. This technique is called confocal laser scanning microscopy (CLSM). By using fluorescent markers of anisometric shape and a polarized laser beam, one can produce 3D images of orientationally ordered materials such as liquid crystals. In this technique, called a fluorescent confocal polarizing microscopy (FCPM), the signal depends on the angle between the transition dipole of the dye and the polarization of probing light. Recent advances in the development of fluorescent markers that can be controllably switched between the bright state and the dark state lead to revolutionary development of far-field optical microscopy with resolution well below the diffraction limit.
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Key References
- Masters, B. R. 2005. Confocal Microscopy and Multiphoton Excitation Microscopy. SPIE Press, Bellingham, Washington.
- An excellent introduction into the principles and applications of confocal and multiphoton microscopy.
- Claxton, N. S., Fellers, T. J., Davidson, M. W. Laser scanning confocal microscopy. Available at http://www.olympusfluoview.com/theory/LSCMIntro.pdf. Accessed November 2, 2011.
- Richly illustrated at-depth discussion of LSCM.
- Prasad, V., Semwogerere, and Weeks, E. R. 2007. Confocal microscopy of colloids. J. Phys. Condens. Matter 19: 113102, 25 pp.
- A comprehensive review of the confocal 3D imaging of colloidal systems.
- Smalyukh, I. I., Shiyanovskii, S. V., and Lavrentovich, O. D. 2001. Three-dimensional imaging of orientational order by fluorescence confocal polarizing microscopy. Chem. Phys. Lett. 336: 88–96.
- A description of the FCPM technique for imaging of the orientationally ordered materials such as liquid crystals.