Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease
diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease
stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of
precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant
clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition
to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for
screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved
with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior
quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide
significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria
and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow
through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was
used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical
virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for
noninvasive in vivo diseases diagnosis.
In this study, photoacoustic imaging is utilized to probe information from oncogene surface molecules of cancer cell with
the aid of specific targeting. The ultimate goal is to provide prediction of clinical outcome and treatment response of
anti-cancer drugs. Different from single targeting in most research, we accomplished multiple targeting to obtain a
molecular profile potentially representing tumor characteristics or to locate the heterogeneous population in one lesion.
By conjugating different antibodies to gold nanorods corresponding to different peak absorption bands, multiple
targeting and simultaneous detection with photoacoustic imaging can be achieved with laser irradiation at the respective
peak optical absorption wavelength. Her2 and EGFR were chosen as our primary target molecules. The targeting
complex was evaluated in two types of oral cancer cells, OECM1 and Cal27. The OECM1 cell line overexpresses Her2
but has low expression of EGFR, while Cal27 cell line expresses both antibodies. Also, the targeting efficacy to OECM1
can be further improved by using mixed nanoprobes. The cancer cells were induced on the back of the mice by
subcutaneous injection. The captured images show that both cancer cells exhibit a higher photoacoustic response
(maximum 3 dB) than control groups with specific targeting, thus demonstrating the feasibility of multiple selective
targeting with bioconjugated gold nanorods. Images of multiple targeting with mixed nanoprobes of OECM1 cells also
reveal further enhancement of targeting (4 dB). The results showed potential of in vivo photoacoustic molecular imaging,
providing a better guidance for diagnosis and treatment of cancer.
Cancer cells presented altered surface molecules to encourage their growth and metastasis. Expression of oncogeneic surface molecules also play important roles in the prediction of clinical outcome and treatment response of anti-cancer drugs. It is thus conceivable that imaging of cancer lesions while simultaneously obtaining their pathogenic information at molecular level of as many oncogenic proteins as possible is of great clinical significance. Gold nanoparticles have been used as a contrast agent for photoacoustic imaging. In addition, gold nanoparticles can be bioconjugated to probe certain molecular processes. An intriguing property of gold nanoparticles is its ability to tailor its optical properties. For
example, size effects on the surface plasmon absorption of spherical gold nanoparticles have shown that the peak optical absorption red-shifts with the increasing particle size. In addition, the optical absorption spectrum of cylindrical gold nanoparticles (i.e., gold nanorods) exhibits a strong absorption band that is directly related to the aspect ratio. With these unique characteristics, selective targeting can be achieved in photoacoustic molecular imaging. Specifically, gold nanorods with different aspect ratios can be bioconjugated to different antibodies. Multiple targeting and simultaneous detection can then be achieved by using laser irradiation at the respective peak optical absorption wavelength. In this study, photoacoustic multiple targeting using gold nanorods is experimentally demonstrated. We have chosen Her2 and CXCR4 as our primary target molecule as Her2 expression is associated with growth characteristics and sensitivity to Herceptin chemotherapy. On the other hand, CXCR4 expression predict the organ-specific metastatic potential of the cancer cells for clinical intervention in advance. Monoclonal antibody (mAb) against Her2/neu was conjugated to nanorods with several different aspect ratios. The agarose gel is suitable for photoacoustic signal acquisition. A wavelength tunable Ti-Sapphire laser was used for laser irradiation and a 1 MHz ultrasound transducer was used for acoustic detection. The optical wavelength of the laser was tuned between 800 nm and 940 nm, corresponding to gold nanorods of an aspect ratio ranging from 3.7 to 5.9. The results clearly show the potential of photoacoustic molecular imaging with multiple targeting in revealing different oncogene expression levels of the cancer cells.