A brief overview of nonlinear microscopy in biomedicine is presented. Some of the main results of the contributions of the Focus Issue are also briefly discussed.
© Optical Society of America
It has been almost a decade since the potential of using nonlinear optical effect for high resolution microscopy imaging was realized . Many of the basic technical issues related to this new technology have been resolved. The resolution of multiphoton microscopes has been measured and verified against theoretical predictions [2,3]. The quantum yields of fluorescent probes suitable for multiphoton microscopy have been determined and new probes optimized for this application are being synthesized [4,5]. In depth comparison between two-photon and confocal microscopy has been performed .
In addition, two-photon microscopy has found novel applications in many areas of biology, biophysics, and medicine. The efficiency of two-photon excitation to discriminate against background fluorescence has allowed the development of two-photon single molecule spectroscopy and two-photon fluorescence correlation spectroscopy [7, 8]. The combination of two-photon excitation with fluorescence spectroscopy enables in vivo monitoring of intracellular biochemistry [9,10,11]. The low tissue absorbancy of infrared light and the non-invasive nature of two-photon excitation has facilitated major advances in embryology studies and noninvasive tissue diagnosis [12–15]. Also see papers by Mohler and White and So et al. in this issue. A combination of confocal reflected light and two-photon fluorescence imaging has been shown to provide complementary structural information in tissues [16, 17, see also Masters in this issue]. Realizing that three- and higher-photon excitation in microscope is useful for imaging deep UV chromophores, three- and high photon microscopy has been developed [18,19,20]. The unique ability of two-photon microscopy to effect chemical reaction in a subfemtoliter volume has been utilized to activate caged compounds with exquisite spatial precision [21,22]. Realizing that the data rate of a typical scanning microscope is too slow for clinical applications or to observe fast kinetics in cells, video rate two-photon microscope systems have been developed [23,24]. The development of multiphoton imaging systems based on a lower cost, picosecond pulse laser or cw lasers may allow wider adoption of this new technology . Finally, realizing that fluorescent labeling of a specimen is sometimes undesirable, nonlinear microscopy based on second and third harmonic light generation has been developed . Also see Squier et al. in this issue.
The technology development in the field of nonlinear microscopy has been exciting. The pace of this innovation is expected to continue but may be at a slower rate as multi-photon imaging reaches maturity. Recently, we have observed that the number of biological and medical researchers adopting multiphoton microscopy as a routine laboratory tool has been increasing rapidly. Hopefully, the next decade will be a time in which the development practice of multiphoton microscopy will come to fruition bringing new scientific insights in biology and improving the practice to medical procedures.
References and links
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