Ovarian Imaging in Animal Models

Two Photon Excited Fluorescence

TPEF imaging is based on a non-linear absorption of photons. Two photons are simultaneously absorbed, allowing for an excited state at an energy level approximately twice the excitation light or half the wavelength with the excitation volume defined by a region where the focused excitation light surpasses the non-linear threshold. The re‐emitted fluorescence is then at lower energy than the excited state but the emission wavelengths are actually less than initial excitation light. TPEF imaging offers some key advantages: a large depth of penetration due to excitation wavelengths in the near-infrared to infrared region, a high resolution limited only by the spatial extent of the intensity threshold above which non linear excitation processes occur, and a low average laser energy deposited in the tissue- less than that provided by a continuous mode laser resulting in less photo‐toxicity in live tissue. Combining these advantages creates a technology suited for live imaging of tissues particularly epithelial tissues. Using TPEF imaging, we can obtain spatially and spectrally resolved images of ovarian autofluorescence, unlike the depth-weighted, point spectroscopy measurements currently made with LIF.

Second Harmonic Generation

Unlike TPEF, SHG is a scattering phenomena rather than absorptive processes. SHG is highly dependent on a non-entrosymmetric environment where the second order polarization term is non-zero, and SHG occurs at half the excitation wavelength as a narrow band compared to the broad fluorescence emitted by TPEF. For example, tissue protein structures such as collagen, myosin, and tubulin exhibit strong SHG properties providing contrast in tissue structure. Specifically, fibrilar collagen (e.g. type I) , which is an important constituent in the ovarian stroma, provides a strong signal. One of the investigators has shown that there are significant differences in the TPEF and SHG signal of ex vivo human ovarian tissue between normal and abnormal samples [Kirkpatrick et al. 2007]. We have performed pilot studies of TPEF and SHG imaging on ex vivo mouse ovaries. En face image and computed cross sections from an image stack (green: TPEF, white: SHG) of a control ovary is shown in figure 4. Typical cell autofluorescence (bright cytoplasm, dark nucleus), primary and secondary follicles and faint SHG from straight ordered collagen are seen. Figure 5 presents images from a VCD/DMBA treated ovary, with a lack of cellular signature, strong SHG from wavy collagen, and hypointense follicular remnants. These initial images suggest that we will be able to see differences in cells and connective tissue in transforming ovary.

Figure 5. En face and computed cross-section images of VCD-DMBA treated mouse ovary, showing TPEF (green) and SHG (white). No ordered cell structure is seen. SHG from collagen is strong and wavy. Small hypointense regions are follicular remnant degeneration.

Figure 4. En face and computed cross-section images of normal mouse ovary, showing TPEF (green) and SHG (white). Hypointense follicles and abundant ordered cells are seen, SHG from collagen is faint and ordered.

References
         Brewer MA, Utzinger U, Barton JK, Hoying JB, Kirkpatrick ND, Brands WR, Davis JR, Hunt K, Stevens SJ, Gmitro AF, “Imaging of the ovary,” Technol Cancer Res Treat. 3(6):617-27, 2004.
         Hariri LP, Bonnema GT, Schmidt K, Winkler AM, Korde V, Hatch K, Brewer M, Barton JK “Laparoscopic optical coherence tomography imaging of human ovarian cancer,” accepted for publication in Gynecologic Oncology, 2009.
         Hoyer PB, Davis JR, Bedrnicek JB, Marion SL, Christian PJ, Barton JK, Brewer MA, "Ovarian neoplasm development by 7,12-dimethylbenz[a]anthracene (DMBA) in a chemically-induced rat model of ovarian failure,” Gynecological Oncology, 112:610615, 2009.
         Kirkpatrick ND, Brewer MA, Utzinger U, “Endogenous optical biomarkers of ovarian cancer evaluated with multiphoton microscopy,” Cancer Epidemiol Biomarkers Prev. 16(10):2048-57, 2007.
         Korde VR, Liebmann E, Barton JK, “Design of a handheld optical coherence microscopy endoscope,” Proceedings of the SPIE 7172:71720D, 2009.
         Skala, M., et al., In vivo Multiphoton Fluorescence Lifetime Imaging of Free and Protein-bound NADH in Normal and Pre-cancerous Epithelia. Optical Society of America: Biomedical Topical Meeting, 2006.