The first in vivo optical virtual biopsy based on epi-third-harmonic-generation (THG) microscopy is successfully demonstrated using Syrian hamster oral mucosa as a model system. Without complex physical biopsy procedures, epi-THG microscopy can provide high spatial resolution dynamic images of oral mucosa and sub-mucosa in all three dimensions. The demonstrated intra-vital epi-THG microscopy provide high resolution observation of blood flow in the capillary and could be a promising tool to image angiogenesis, which is an important feature for many human diseases including malignancies. The system setup of epi-THG microscopy can be easily integrated with other nonlinear optical microscopy such as second-harmonic generation and multi-photon fluorescence microscopy by using the same laser system to provide better integrated molecular and structural information for future clinical diagnosis. By adding 6% acetic acid solution on the mucosa, THG contrast on the borders of nuclei was found to be greatly enhanced due to the alterations of their linear and nonlinear THG susceptibilities. With a virtual-transition-based technology without using fluorescence, the optical epi-THG biopsy we demonstrated shows promise for future noninvasive in vivo diseases examinations.

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.

Harmonics-based optical microscopy has been widely applied in biomedical researches due to its noninvasiveness to the studied biomaterials. Due to momentum conservation consideration, most previous studies collect harmonics generation signals in a forward geometry, especially for third harmonic generation signals. However, the adopted forward transmission type geometry is not feasible for future clinical diagnosis. In this paper, first virtual biopsy based on backward propagating optical higher harmonics, combining second harmonic and third harmonic, is demonstrated in the fixed human skin specimens. In our study, third harmonic generation can provide morphologic information including the distribution of basal cells while second harmonic generation can provide distribution of collagen fibers in dermis. Therefore, this technique is ideal for future noninvasive in vivo skin disease examination without dye.

This paper reports a facile and effortless method to realize three-dimensional (3D) molecular third-harmonic-generation (THG) microscopy through the technique of resonance enhancement with absorbing dye. Hematoxylin, a popular absorbing stain, is applied as an example to verify the multiphoton resonant enhancement based on the 1230 nm excitation light and can selectively enhance THG yield at cell nuclear sites in the studied specimens, serving as a cell nucleus contrast agent. It is concluded that combining THG microscopy with the mature staining technique can readily achieve 3D molecular imaging without using fluorescence.

Received October 14, 2005; revised January 7, 2006; accepted January 9, 2006; posted January 12, 2006 (Doc. ID 65391) The two-photon excitation action cross section of Hc-Red fluorescent proteins (Hc-RFPs) is measured and found to be of the same order as that of enhanced green fluorescent proteins. With a 618 nm emission wavelength in the far-red region and with an excitation wavelength around 1200 nm, Hc-RPF-based two-photon fluorescence microscopy (2PFM) can offer deep penetration capability inside live samples and is ideal for in vivo gene expression study and biomolecular imaging in live objects. In vivo 2PFM of the developing heart deep inside a transgenic zebrafish embryo tagged by Hc-RFP is also successfully demonstrated.