2.1.

Mouse Models of Lung Adenocarcinoma

LSL-K-ras G12D mice²⁵ were obtained from the Mouse Models of Human Cancer Consortium and backcrossed either onto a pure 129SvEv or onto an FVB/N genetic background. To induce Cre-mediated recombination and K-ras G12D activation in the lungs, adult mice were anesthetized with 2.5% Avertin (tribromoethanol and tert-amyl alcohol) and were administered $6.7\times {10}^8$ plaque-forming units (PFU) of Ad-Cre virus, diluted in 40 μl of phosphate buffered saline (PBS), either intratracheally or intranasally. Note that $1:10$, $1:100$ or $1:1000$ dilutions were used for some mice. The lungs were harvested 6 to 20 weeks post infection from euthanized mice. $\mathrm{Rrm}{2}^{\mathrm{Tg}}$ mice were maintained on pure FVB/N genetic background as previously described.²⁶ All animal housing and experimentation was performed in accordance with institutional animal care and use guidelines.

2.2.

Lung Tissue Processing and Histopathological Assessment

Mice were euthanized by carbon dioxide asphyxiation. The lung lobes were removed and visually inspected for abnormalities. Normal lung has a uniform pink color throughout the surface of the lobes, whereas regions with potential abnormalities appear as white or red discolorations in the smooth surface of the lung. Sites with abnormal appearance were selected on each lobe and imaged within two hours after euthanasia. All lobes were kept in chilled PBS until imaging. Immediately after imaging, the lobes were fixed in 10% buffered formalin or 4% paraformaldehyde. H&E stained sections were prepared from each lobe following standard embedding and staining protocols. H&E sections ($\sim 6\mu \mathrm{m}$ thick) were obtained from a similar depth and location within samples to that imaged by MPM. Low-magnification micrographs were obtained for each H&E section and compared to a low-magnification MPM image of the lobe prior to tissue fixation. Using structural features present on both the H&E and MPM image, the location of the site imaged at high-magnification MPM was approximated on the H&E section. Thus, histopathological assessment was performed at a site on the H&E section comparable to the site imaged with MPM prior to tissue processing. Pathological assessment was performed according to guidelines in the Mouse Models of Human Cancers Consortium.²⁷

Lung samples were also obtained from a 12-year-old female spayed Miniature Poodle treated at the Cornell University Hospital for Animals for bronchioloalveolar carcinoma of the left caudal lung lobe. The left caudal lung lobe was removed by standard lung lobectomy by a veterinary surgeon. Clinically abnormal and normal samples were obtained from the resected lobe by a veterinary oncologist. Prior to surgery, an informed consent form was signed by the owner of the dog. The samples were kept in chilled PBS for about two hours prior to imaging. Similar tissue imaging and processing were followed as for mouse lungs.

2.3.

MPM of Unprocessed Lung Tissue

Imaging was performed with a commercial multiphoton microscope (Olympus Fluoview® FV1000MPE, Olympus America Inc, Center Valley, PA) at a shorter (760 nm) and longer (860 nm) excitation. An Olympus $25\times$, 1.05 NA water immersion objective was used and all images were obtained with $\sim 23\mathrm{mW}$ (at 760 nm excitation) and $\sim 25\mathrm{mW}$ (at 860 nm excitation) average power at the objective, pixel dwell time of 2 μs and at $1.1\sec /\text{frame}$. Displayed MPM images represent a Kalman average of three frames. Autofluorescence and SHG were captured by three detection channels, equipped with multi-alkali photomultiplier tubes. The first channel detected signal at 420 to 460 nm, the second and third channels captured endogenous fluorescence at 500 to 550 nm and at 575 to 635 nm; respectively. Images from each channel were pseudocolored and superimposed. Displayed MPM images represent superimposed images: “green” pixels indicates fluorescence detected predominantly in the second channel, “red” indicates fluorescence detected predominantly in the third channel and “blue” indicates emission at 420 to 460 nm (including SHG at 860 nm excitation only). The contrast and brightness were adjusted equally for all displayed MPM images at a particular excitation wavelength.

MPM was performed on lobes from eight control mice, including uninfected and Ad-Cre infected wild-type mice, and on lobes from 10 Ad-Cre infected LSL-K-ras G12D mice and two tumor bearing $\mathrm{Rrm}{2}^{\mathrm{Tg}}$ mice. Images were taken from 1 to 4 sites per lobe. Unaffected lobes with normal appearance were imaged approximately 20 to 30 μm below the pleura at a representative site. Lobes with grossly visible abnormalities were imaged at several sites $\sim 20$ to 30 μm below the pleura; an image was taken from the center of each nodule and the tissue surrounding the nodule. For larger or heterogeneous nodules, images were taken from different areas within the nodule. In total, images were obtained from eleven tumor free unaffected (normal) lung sites, four adenomas, six adenocarcinomas, three sites with focal inflammation and six regions with diffuse hyperplasia. MPM images from adenomas and adenocarcinomas were analyzed by determining the Green (500 to 550 nm)/Blue (420 to 460 nm) fluorescence emission ratio for each MPM image (defined as the average pixel intensity value from the green channel (500 to 550 nm) divided by the sum of the average pixel intensity values from the blue (420 to 460 nm) and green channel (500 to 550 nm). The ratio was calculated only for pixels within the nodule and not from the surrounding tissue.

2.4.

Two-Photon Excited Microspectroscopy of Unprocessed Lung Tissue

Microspectroscopy was performed using a spectrograph (Andor Technology, South Windsor, CT, USA) coupled to the microscope with a multimode optical fiber. A spectroscopic EEM CCD (Andor Technology, South Windsor, CT, USA), attached to the spectrograph, was used in full vertical binning mode to measure spectra from the imaged sample. Spectra were obtained by scanning a zoomed in ($73\times 73\mu {\mathrm{m}}^2$) region within each MPM image for an exposure time of 1.18 sec and average power at the objective of $\sim 23\mathrm{mW}$ (at 760 nm excitation) and $\sim 25\mathrm{mW}$ (at 860 nm excitation). Spectra were obtained at $\sim 20$ to 30 μm below the pleura. No photodamage was observed in the zoomed-in regions after spectroscopic acquisition.

Microspectroscopy was performed on lesions with dense cellular aggregations such as adenomas, adenocarcinomas and focal inflammatory sites. During MPM imaging at 760 nm, regions within each tumor composed of cells with dominant fluorescence at 420 to 460 nm were visually selected. Next, three spectra were sampled from zoomed in ($73\times 73\mu {\mathrm{m}}^2$) areas within these preselected regions. In focal inflammatory sites, clusters of inflammatory cells with yellow appearance on MPM images were sampled. Spectra were also obtained from normal sites by sampling zoomed in regions of the alveolar septa from unaffected alveoli. All spectra were background subtracted, smoothed by a Savitzky-Golay filter (MATLAB), and normalized by the maximum intensity value in the 400 to 650 nm region prior to analysis.

3.1.

Two-Photon Excited Intrinsic Fluorescence Features of Tumor Free Peripheral Mouse Lung

To demonstrate the ability of MPM to differentiate normal from diseased lung, ex vivo MPM was first performed on tumor-free unaffected peripheral lung [Figs. 1(a)–1(f)]. MPM images showed similar microanatomical features to histopathology, with intact alveolar spaces and clearly apparent septa. The thin layer of epithelial cells comprising the alveoli was not visualized by MPM, and the two-photon excited intrinsic emission appeared to originate from the alveolar septa, not the associated epithelium. At 760 nm excitation, the brightest fluorescence originated from granular structures; individual cells were not readily visualized. Two-photon excited fluorescence spectra of intact alveoli from eleven different tumor-free unaffected lung sites peaked at $\sim 480\mathrm{nm}$ (data not shown), which differed from fluorescence of typical cellular fluorophores such as NADH and flavins.^12,17,18 At 860 nm excitation, fluorescence originated predominantly from fibers within the alveolar walls that appeared green in MPM images [Fig. 1(c)], indicating fluorescence detected at 500 to 550 nm. Some alveoli also displayed fibers with blue appearance in MPM images [Figs. 1(f) and 2(c)], probably caused by SHG at 430 nm from collagen fibers in the alveolar septa. Microanatomically, this signal correlates well with the collagen distribution in Masson’s trichrome stained tumor-free peripheral lung [Fig. 2(a)], indicating that fibers with blue appearance at 860 nm represent collagen from the alveolar septa. Intact alveoli without a readily visualized cellular component were observed in MPM images from all tumor-free lung sites and used as an MPM-detected feature to differentiate tumor-free and diseased lung in this study (Table 1).

Fig. 1

Multiphoton microscopy (MPM)-detected intrinsic fluorescence characteristics of normal, inflammatory and hyperplasic peripheral mouse lung at shorter (760 nm) and longer (860 nm) wavelength excitation. (a–c), Images from an uninfected wild-type control mouse. H&E image displays normal lung parenchyma composed of alveolar spaces separated by thin septa. MPM images enable detection of intact alveolar spaces and septa and minimal cellular infiltration in the lumens. (d–f), Images from an Ad-Cre infected wild-type control mouse. H&E image displays normal lung parenchyma with a few small macrophages within alveolar spaces. (g–i), A site with diffuse inflammation. H&E image shows alveolar spaces with alveolar macrophages with intensely eosinophilic cytoplasm. MPM detects cells within alveolar lumina with yellow appearance, indicating fluorescence detected at 500 to 635 nm, channel 2 and 3. (j–l), A site with focal inflammation. H&E image shows perivascular spaces expanded by infiltrates of small lymphocytes, and alveolar spaces with eosinophilic macrophages. MPM detects cellular aggregates with bright fluorescence at detected at 500 to 635 nm. (m–o), A site with diffuse hyperplasia. H&E image shows accumulation of oval to cuboidal epithelial cells with no compression of the adjacent parenchyma. MPM imaging detects the accumulation of cells. At 760 nm excitation, these cells appear blue, indicating fluorescence detected predominantly at 420 to 460 nm, channel 1, while at 860 nm excitation they appear dim (fluorescence with very weak intensity). All samples were imaged before (MPM) and after (H&E) tissue processing. Scale bar: 100 μm.

Fig. 2

Masson’s trichrome (a), Verhoeff stain (b) and multiphoton microscopy (MPM) at 860 nm (c) of a tumor-free peripheral lung site comparing the location of collagen and elastin fibers in alveolar septa before (c) and after (a, b) tissue processing. Collagen fibers appear blue in (a), whereas elastin fibers appear black in (b). Inset: higher power view of collagen (a) and elastin (b) fibers. In the MPM image, fibers with dominant emission at 420 to 460 nm (blue color) are seen in the pleura and along alveolar septa. This signal is most likely due to SHG (at 430 nm) from collagen fibers. Note that the pattern of the collagen distribution shown in the Masson's trichrome image is very similar to the distribution fibers with blue color in the MPM image. Scale bar: 100 μm.

Table 1

Structural and spectral features observed in multiphoton microscopy (MPM) images of normal, inflammatory and neoplastic lung. The spectral features observed in MPM images were also supported by two-photon excited microspectroscopy. While standard histopathological criteria were used to diagnose the lung samples after tissue processing, the summarized features were based on observation of MPM images obtained from fresh, unprocessed tissue.

Next, we evaluated the two-photon excited fluorescence patterns in MPM images from lung sites with diffuse and focal inflammation [Figs. 1(g)–1(l)]. Sites with diffuse inflammation showed readily identifiable individual cells infiltrating the alveolar lumina, whereas focal inflammations appeared as denser cellular accumulations without clearly visualized borders. Histopathology indicated that the cellular infiltration in the diffuse inflammation site was caused by macrophages, whereas the focal inflammatory lesion was composed of lymphocytes and macrophages. The cells in both the diffuse and focal inflammation site appeared yellow in MPM images, indicating fluorescence detected at longer wavelengths (500 to 635 nm, channel 2 and 3). Furthermore, two-photon excited emission spectra from groups of cells within the focal inflammation showed a pronounced red-shifted emission at both 760 and 860 nm excitation [Figs. 3(a) and 3(b)]. Denser cellular accumulations with intrinsic fluorescence emission at longer wavelengths were observed in MPM images from all focal inflammation sites and used as an MPM-detected feature to differentiate focal inflammatory sites and neoplastic cellular masses in this study (Table 1). Infiltration of cells fluorescing at longer emission wavelengths was also observed in the lung parenchyma next to most of the adenocarcinomas. Histopathological assessment indicated that the alveoli next to most adenocarcinoma were filled with macrophages with eosinophilic cytoplasm.

Fig. 3

Two-photon excited microspectroscopy illustrating differences in the intrinsic cellular fluorescence of mouse adenomas, adenocarcinomas and focal inflammations at shorter (760 nm) and longer (860 nm) wavelength excitation. (a–b), Representative spectra obtained from group of cells within an adenocarcinoma (blue lines), an adenoma (cyan lines) and a focal inflammation site (red lines). To display the spectral variation within each nodule, three separate spectra were recorded per site. All spectra were normalized by their maximum intensity value. (c–d), Average spectra from six different adenocarcinomas, four adenomas, and three focal inflammations. The error bars represent one standard deviation of the average fluorescence intensity values at emission wavelengths in the 400 to 620 nm region. (e–f), Selected emission wavelength region from the average spectra in (c) and (d) emphasizing that spectral differentiation between adenomas and adenocarcinomas is achieved at 760 nm and not 860 nm excitation.

3.2.

MPM of Diffuse Hyperplasia in Peripheral Mouse Lung

Precursor lesions, such as AAH, in the LSL-K-ras G12D lung adenocarcinoma model were previously described as an accumulation of atypical epithelial cells on alveolar walls.²⁵ MPM images from peripheral lung with diffuse hyperplasia detected the accumulation of oval to cuboidal epithelial cells on alveolar walls (as confirmed by histopathology) without compression of the adjacent alveoli [Figs. 1(m)–1(o)]. At 760 nm excitation, these cells appeared blue in MPM images, indicating fluorescence detected predominantly at 420 to 460 nm. Furthermore, two-photon excited spectra from these cells displayed an emission peak at $\sim 455\mathrm{nm}$ (data not shown), which is similar to emission maxima of spectra from the mouse tumors [Figs. 3(a) and 3(c)] and to values cited by previous publications on MPM of unprocessed tissue and attributed to cellular NADH.^17,18

3.3.

MPM of Mouse Lung Neoplasms Such as Adenomas and Adenocarcinomas

To elucidate the two-photon excited endogenous patterns of mouse lung neoplasms, we first imaged small adenomas (Fig. 4). In the H&E images, the adenomas appeared as well-demarcated nodules composed of neoplastic cuboidal epithelial cells that obliterated the alveolar spaces. MPM at 760 nm detected similar microanatomical patterns; the nodules appeared as dense cellular masses completely distorting the lung parenchyma, while the tissue immediately next to the nodule had intact alveoli. At 760 nm excitation, the majority of cells within the adenomas appeared blue in the MPM images, indicating fluorescence detected predominantly at 420 to 460 nm. Microspectroscopy confirmed that the two-photon excited fluorescence spectra from groups of cells within the adenomas peaked at $\sim 455\mathrm{nm}$ [Figs. 3(a) and 3(c)]. Dense cellular aggregates with intrinsic fluorescence at 420 to 460 nm were observed in MPM images from all adenomas and described here as an MPM-detected feature (Table 1). At 860 nm excitation, the adenomas displayed an increased presence of fibers with dominant emission at 420 to 460 nm, as compared to the surrounding lung parenchyma [Figs. 4(c) and 4(f)]. This feature was visualized in three out of four adenomas. The adenoma that did not clearly display this feature was surrounded by lung parenchyma with diffuse hyperplasia.

Fig. 4

Multiphoton microscopy (MPM)-detected intrinsic fluorescence characteristics of mouse adenomas (a type of benign lung tumor). (a–c), A papillary adenoma. The H&E image shows a well-demarcated nodule within the lung parenchyma composed of neoplastic cuboidal epithelial cells arranged in papillary projections. MPM at 760 nm shows a compact mass with cells, most of which have blue appearance (fluorescence detected at 420 to 460 nm). MPM at 860 nm enables the detection of more fibers with blue appearance within the mass compared to the surrounding alveoli. (d–f), A solid adenoma; the H&E image shows a well-demarcated nodule composed of neoplastic cuboidal cells, arranged in closely packed cords that obliterate the alveolar spaces. MPM detects similar patterns as (a–c). Scale bar: 100 μm.

MPM images from a mouse lung adenocarcinoma are shown in Fig. 5. At 760 nm excitation, most of the cells from a site within the tumor displayed dominant fluorescence emission at 420 to 460 nm, similar to the adenomas. More complex MPM patterns were observed at the periphery of the tumor [Fig. 5(h)] and the surrounding lung parenchyma [Fig. 5(k)] compared to the adenomas. In particular, the periphery of the lesion appeared to have cells with diverse structural and spectral patterns, while the surrounding lung parenchyma had marked infiltration of cells fluorescing at longer wavelengths. Histopathology confirmed the presence of focally extensive infiltrates of lymphocytes mixed with macrophages at the periphery of the lesion and marked macrophage infiltration in the surrounding alveoli. All adenocarcinomas in this study appeared as dense cellular masses in MPM images, and most of the cells within these masses displayed dominant cellular fluorescence emission at 420 to 460 nm. Therefore, the detection of cellular aggregates with dominant intrinsic fluorescence at 420 to 460 was described here as an MPM-detected characteristic of mouse adenocarcinomas (Table 1). At 860 nm excitation, the site within the adenocarcinoma [Fig. 5(f)] displayed more fibers with dominant emission at 420 to 460 nm, compared to the surrounding lung parenchyma [Fig. 5(l)]. Similar to the adenomas, this feature was detected by MPM at 860 nm in most (four out of six) adenocarcinomas and is likely to be SHG from collagen fibers at 430 nm. One of the adenocarcinomas that did not clearly display this feature was surrounded by lung parenchyma with diffuse hyperplasia. The biological basis for this feature has not been determined; however, advanced mouse lung adenocarcinomas are reported to deposit desmoplastic stroma around tumor cells²⁸ and the observed fiber accumulation by MPM could signify increased stromal (fibrous) deposition.

Fig. 5

Images of a mouse adenocarcinoma illustrating differences in multiphoton microscopy (MPM)-detected characteristics at sites within (site 1), at the periphery (site 2) and next to the tumor (site 3). (a–c), Low-magnification images indicating the approximate location of the sites displayed in (d–l). Histopathological assessment (a) reveals a well-demarcated, expansile neoplastic mass composed of epithelial cells that form papillary projections and solid areas and efface the pulmonary parenchyma. (d–f), A site within the tumor. Histopathologically (d), the mass consists of neoplastic cells with loss of nuclear polarity, variation in nuclear size and occasional mitotic figures (inset). MPM at 760 nm (e) detects a compact mass of cells; most of these cells appear blue (fluorescence detected at 420 to 460 nm, channel 1). (g–i), A site at the periphery of the tumor. The H&E shows focally extensive infiltrate of lymphocytes mixed with eosinophilic macrophages. MPM at 760 nm detects clusters of cells with different spectral patterns. (j–l), Lung parenchyma next to the mass. In the H&E image (j), the alveolar spaces are filled with numerous, variably-sized, eosinophilic macrophages. MPM at 760 nm detects cells with yellow appearance (fluorescence at 500 to 630 nm, channel 1 and 2) infiltrating the alveolar lumens. Scale bar: (a–c) 1000 μm, (d–l) 100 μm.

Visual observation of MPM images at 760 nm showed that most of the cells within the adenomas and adenocarcinomas displayed dominant fluorescence emission at 420 to 460 nm. However, quantitative analysis of the MPM images revealed that, compared to cells within adenomas, cells within the adenocarcinomas displayed lower fluorescence emission at 500 to 550 nm, relative to fluorescence emission at 420 to 460 nm. In particular, we calculated the Green (500 to 550 nm)/Blue (420 to 460 nm) emission ratio (the average pixel intensity from the 500 to 550 nm channel vs. the average pixel intensity from the 420 to 460 nm channel) from MPM images of all adenomas and (the central regions) of all adenocarcinomas. On average, at 760 nm excitation, the cells within adenocarcinomas displayed a Green (500 to 550 nm)/Blue (420 to 460 nm) ratio that was lower than the adenomas (ratio for adenocarcinomas: $0.38\pm 0.02$, $\text{mean}\pm \mathrm{SD}$, $n=6$; ratio for adenomas: $0.45\pm 0.04$, $\text{mean}\pm \mathrm{SD}$, $n=4$). This indicates that, for the current set of MPM images, cells within the malignancies displayed a less pronounced fluorescence at 500 to 550 nm (blue-shifted fluorescence) compared to the benign tumors.

3.4.

Spectroscopic Evaluation of Cells Within Mouse Lung Neoplasms and Focal Inflammatory Sites

To further elucidate differences in cellular fluorescence patterns of lung neoplasms and focal inflammations, we collected two-photon excited spectra from groups of cells from an adenoma, adenocarcinoma and focal inflammation [Fig. 3(a)]. At 760 nm excitation, cells from the adenocarcinoma showed similar emission at 400 to 460 nm, but less pronounced spectral broadening at 500 to 600 nm, compared to those from the adenoma. Cells from the focal inflammation showed a marked red-shifted fluorescence compared to cells from either the adenoma or adenocarcinoma, especially at longer emission wavelengths (500 to 640 nm). To determine the degree of variations between samples, average two-photon excited emission spectra were calculated from multiple samples for each histopathological category [Fig. 3(c)]. At 760 nm excitation, average spectra from the adenocarcinomas and adenomas showed similar cellular fluorescence at 400 to 460 nm, but different spectral emission at the 500 to 600 nm, confirming spectral trends observed from individual samples [Fig. 3(a)]. At 860 nm excitation, spectra from adenomas and adenocarcinomas displayed a similar emission peak and shape, whereas spectra from focal inflammations displayed a distinct red-shifted spectral broadening compared to all other categories. Spectroscopic trends summarized in Fig. 3 demonstrate that for the current data set, spectral differentiation between adenomas and adenocarcinomas was best achieved at 760 nm (and not at 860 nm) excitation and at 500 to 600 nm emission. However, focal inflammations were spectrally differentiated from lung neoplasms at both 760 and 860 nm excitation.

3.4.1.

MPM of canine lung carcinoma

To confirm that our results were reproducible in spontaneously occurring lung carcinomas and applicable to larger mammals, we imaged normal and diseased lung tissue from a dog treated at Cornell University Hospital for Animals for bronchioloalveolar carcinoma of the left caudal lung lobe (Fig. 6). MPM images from an abnormal region with bronchioloalveolar carcinoma showed similar microanatomy to the H&E images, with cellular projections completely displacing the lung parenchyma. At 760 nm excitation, the cellular projections appeared blue in MPM images [Figs. 6(b) and 6(e)] and displayed emission spectra with a peak at $\sim 455\mathrm{nm}$ [Fig. 6(m), blue lines]. MPM images from another region within the abnormal sample displayed accumulation of cells with yellow appearance (fluorescence at 500 to 635 nm). This region was histopathologically confirmed as cancer-associated inflammation. At 760 nm excitation, emission spectra from these cells displayed a red-shifted fluorescence compared to cells within the carcinoma region [Fig. 6(m), red lines]. By comparison, MPM of tumor-free peripheral lung detected intact alveoli without readily visualized cellular component. These features were similar to MPM detected characteristics of mouse normal, inflammatory and malignant lung and suggest that the MPM detected characteristics of mouse neoplasms (Table 1) would be also valid for spontaneously occurring lung carcinomas in larger mammals.

Fig. 6

Images from a resected canine lung lobe with naturally occurring bronchioloalveolar carcinoma before multiphoton microscopy (MPM) and after (H&E) tissue processing. (a–f), A site within the carcinoma displaying cellular papillary projections that compress the surrounding parenchyma. (g–i), A site histopathologically confirmed as infiltrates of inflammatory cells (macrophages and lymphocytes) associated with the tumor. (j–l), A site from an area of normal, tumor-free pulmonary parenchyma. (m), Two-photon excited fluorescence spectra at 760 nm excitation from cells within the carcinoma site (blue lines) and the cancer-associated inflammation site (red lines), and from intact alveoli in the tumor-free lung site (green lines). Scale bar: 100 μm.