Multiphoton microscopy as a diagnostic imaging modality for lung cancer

Abstract

Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on two-photon excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

Figure 1

MPM imaging of tumor-free murine lung showing normal alveolar structure. The sample was collected from an AdenoCre infected wild-type control mouse from the LSL-Kras G12D model. After imaging, the sample was fixed in 4% paraformaldehyde, and embedded, sectioned, and stained with H&E (A). (B) Low-magnification MPM image showing the overall morphology of the sample. Note that the MPM and H&E image are from comparable but not identical regions. (C) High-magnification MPM image showing individual alveoli. Two-photon fluorescence and SHG were excited at 780 nm and detected at 355-425 nm, 440-500 nm and 505-655 nm emission bands. The displayed MPM images were generated by superimposing the individual images from the three bands; blue color indicates dominant emission at 355-425 nm; green color represents fluorescence in the 440-500 nm region. Scale bar: 100um.

Figure 2

MPM imaging of lipid pneumonia in murine lungs. The sample was collected from a 14 month old Rrm2^Tg mouse with a small white mass visible on the lung surface at necropsy. After imaging, the sample was fixed in 10% buffered formalin, and then embedded, sectioned, and stained with H&E (A). A series of MPM images was collected and assembled into a mosaic composite (B). The MPM and H&E images are from similar but not identical regions of the sample. Two-photon fluorescence and SHG were excited at 780 nm and detected at 355-425 nm, 440-500nm and 505-655 nm emission bands. In the displayed superimposed images blue color indicates emission at 355-425 nm; green color represents fluorescence at 440-500 nm; and red color indicates fluorescence at 505-655 nm. The black arrow in (A) points to a lipid-filled macrophage, characteristic of lipid pneumonia in mice. Scale bar: 100um.

Figure 3

MPM imaging of a benign lung adenoma. The sample was taken from an 8.5 month old Rrm2^Tg mouse. A white mass was visible on the lung surface at necropsy (A) and was identified as an adenoma in H&E sections imaged at high- (B) or low- (D) magnification. MPM and H&E images were generated at the center of the adenoma (B,C) or at an adjacent non-neoplastic site (E,F) with marked macrophage infiltration. The MPM (C,F) and H&E (B,E) images are from similar but not identical regions of the sample. The black arrow in (A) points to the location of the adenoma. The scale bar: 5000um in (A) and 100um in (B-F).