High‐resolution simultaneous three‐photon fluorescence and third‐harmonic‐generation microscopy

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. Microsc. Res. Tech. 66:193–197, 2005. © 2005 Wiley‐Liss, Inc.