In comparison with single-photon confocal imaging, TPE fluorescence microscopy reduces overall photobleaching and photodamage by limiting it to the narrow region around the focal plane.1 Using near-infrared TPE fluorescence microscopy, Squirrell et al were able to monitor the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 hours without jeopardizing developmental competence. By contrast, confocal imaging for only 8 hours severely compromised development.71
However, peak light intensities, such as occurring during pulsed illumination, may facilitate photobleaching or photodamage in the narrow region around the focal plane.14,15,17 Koester et al14 and Hopt et al15 concordantly found that at low average excitation light levels, a two-photon absorption process is mainly responsible for cumulative photodamage, but higher order mechanisms will become important at larger excitation intensities. These findings suggest that, at low excitation intensities, fluorescence signal can be obtained only at the expense of photodamage. As a consequence, when imaging over a longer time, laser power should be carefully adjusted to minimize photodamage, but maintain signal-to-noise ratios that are practical for laser scanning fluorescence microscopy.
On the other hand, the damaging potential of high-intensity, two-photon illumination has been successfully exploited to reproducibly generate reactive oxygen species in a small volume fraction of an isolated cardiomyocyte and to subsequently study their effects on cellular metabolism.70 Tirlapur and Konig were able to transfer foreign DNA with great efficiency into a variety of cell types following localized perforation in the cell membrane using femtosecond, high-intensity, near-infrared laser pulses.72
The combined benefits of TPE fluorescence microscopy, including deeper penetration depth in thick, highly scattering biological specimens, and decreased overall fluorophore photobleaching and photodamage compared with confocal microscopy, are particularly useful for developmental studies that require the maintenance of viability to detect sequential events in the same specimen over extended periods of time. Thus, TPE laser scanning microscopy should enable numerous long-term imaging studies in living embryos to reconstruct dynamic processes such as gene and protein expression patterns in three dimensions6 and should enable imaging of three-dimensional cell migration. The advantages and disadvantages of TPLSM, however, for long-term imaging of developmental processes have to be compared with those of a novel optical sectioning technique, named selective plane illumination microscopy,73 which appears to generate even less photodamage/photobleaching than TPLSM at comparable spatial resolution.
Future Developments
Two-photon microscopy excels at high-resolution imaging hundreds of microns deep in living tissue. The maximal imaging depth attainable with TPE is likely to increase even further in the future with the development of chromophores with higher TPE cross sections and implementation of techniques that will reduce fluorescence collection loss caused by absorption and scattering. The development of femtosecond lasers that are tunable to wavelengths in the 1000- to 1300-nm range will further improve laser-beam penetration depth and will enable TPE of longer wavelengths dyes and fluorescent proteins but, at the same time, lower the risk of photobleaching and photodamage. New signal processing algorithms will offer the opportunity to simultaneously image multiple colored, spectrally overlapping markers in living cells and tissue. Finally, the combination of TPE with time-resolved techniques including fluorescence lifetime imaging (which may be useful for analyzing FRET between engineered proteins), fluorescence anisotropy, and fluorescence correlation spectroscopy will provide new opportunities for studying cellular microenvironments and the behavior of fluorescent probes in the intracellular milieu.74,75 Thus, TPE microscopy will remain a valuable tool for imaging cellular and subcellular events within living tissue and, therefore, provide a technical basis for a much-needed integrative approach to biological problems.
Acknowledgments
This work was supported by the NIH. I thank Drs L.J. Field, M.H. Soonpaa, H. Nakajima, W. Zhu, and R. Prabakhar for comments.
Footnotes
Original received September 22, 2004; revision received November 1, 2004; accepted November 2, 2004.
Answers to all your Biology Questions
