## Abstract Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode‐locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO
High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy
✍ Scribed by Shi-Wei Chu; Shih-Peng Tai; Chia-Lin Ho; Chi-Hung Lin; Chi-Kuang Sun
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 170 KB
- Volume
- 66
- Category
- Article
- ISSN
- 1059-910X
No coin nor oath required. For personal study only.
✦ Synopsis
In recent years, nonlinear laser scanning microscopy has gained much attention due to its unique ability of deep optical sectioning. Based on our previous studies, a 1,200-1,300-nm femtosecond laser can provide superior penetration capability with minimized photodamage possibility. However, with the longer wavelength excitation, three-photon-fluorescence (3PF) would be necessary for efficient use of intrinsic and extrinsic visible fluorophores. The three-photon process can provide much better spatial resolution than two-photon-fluorescence due to the cubic power dependency. On the other hand, third-harmonic-generation (THG), another intrinsic three-photon process, is interface-sensitive and can be used as a general structural imaging modality to show the exact location of cellular membranes. The virtual-transition characteristic of THG prevents any excess energy from releasing in bio-tissues and, thus, THG acts as a truly noninvasive imaging tool. Here we demonstrated the first combined 3PF and THG microscopy, which can provide three-dimensional high-resolution images with both functional molecule specificity and sub-micrometer structural mapping capability. The simultaneously acquired 3PF and THG images based on a 1,230-nm Cr:forsterite femtosecond laser are shown with a Hoechst-labeled hepatic cell sample. Strong 3PF around 450 nm from DNA-bounded Hoechst-33258 can be observed inside each nucleus while THG reveals the location of plasma membranes and other membrane-based organelles such as mitochondria. Considering that the maximum-allowable laser power in common nonlinear laser microscopy is less than 10 mW at 800 nm, it is remarkable that even with a 100-mW 1,230-nm incident power, there is no observable photo damage on the cells, demonstrating the noninvasiveness of this novel microscopy technique.
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