2.1.

Sample Preparation

All animal experiments were approved and carried out in accordance with the regulations set forth by the Massachusetts General Hospital Subcommittee on Research Animal Care. Three adult Sprague-Dawley rats (approximately 350 g) were weighed and anesthetized with an intramuscular injection of Ketamine-Xylazine. Once a surgical plane of anesthesia was reached, the abdominal aorta was transected to exsanguate the animals. The trachea was then exposed and catheterized with an 18 G catheter. The lungs were instillation fixed in situ at 20 cm ${\mathrm{H}}_2\mathrm{O}$ pressure through a gravity feed system using a modified Heitzman solution consisting of 10% formaldehyde solution (Fisher Scientific), 10% ethanol (Fisher Scientific), 25% polyethylene glycol 400 (Post Apple Scientific), and 55% laboratory distilled water.¹⁶ After 30 min, the lungs were carefully excised and then immersed within the Heitzman solution while connected to the feed system at the same inflation pressure for a minimum of 48 h. The lungs were then disconnected from the fixation apparatus and air-dried for a minimum of 2 days in a 60°C drying oven while pressurized with air at 20 cm ${\mathrm{H}}_2\mathrm{O}$. Finally, the dry inflated lungs were cut into cylindrical sections, approximately 3 mm in diameter and 1 to 2 mm thick, immersed in PBS and degassed for imaging. After 3-D images were obtained with all three imaging modalities on the same sample, each was placed in formalin and the entire sample was processed for paraffin-embedded histopathology by using standard methods. Five micrometer thick Hematoxylin and eosin (H&E) histopathology slices were obtained, and slides were digitized by using a full-slide scanner (ScanScope CS, Aperio Technologies, Inc, Vista, Calif).

2.2.

Imaging Techniques

Three different optical reflectance-imaging technologies were used to obtain the same approximate field of view within each sample. First, OFDI and SECM images were acquired with a 200 μm thick cover glass between the sample and the focusing optics, and then the cover glass was removed and the sample was imaged with FFOCM. In this section, we describe the basic functionality and system components of each technique. The characteristics of each imaging modality are summarized in Table 1.

Table 1

OFDI, SECM, and FFOCM system parameters used to image PBS-immersed rat lung tissue.

3.1.

OFDI

Figure 1(a) shows a typical cross-sectional OFDI image of a rat lung slice, where the alveoli appear as dark gaps within the highly scattering tissue under the cover glass. Figure 1(b) shows the orthogonal en-face image from the sample with the corresponding H&E histology section shown in Fig. 1(c). Single alveoli can be easily distinguished within the OFDI images up to a depth of approximately 500 μm and the presence of the fixed blood vessels (marked with a V) does not appear to decrease the overall imaging depth significantly. The alveolar wall thickness appears overestimated ($25\pm 6\mathit{\mu }\mathrm{m}$ full width at half maximum (FWHM), $\text{mean}\pm \text{standard deviation}$, $n=31$) compared with the generally accepted wall thickness of a rat alveolus (7 to 10 μm).

Fig. 1

OFDI and H&E histology images of PBS-immersed fixed rat lung slice. (a) Axial cross-section and (b) en-face OFDI images. (c) Corresponding H&E histology sectioned in en-face plane. V indicates a vessel within the tissue. Scale bars, 500 μm.

3.2.

SECM

Figure 2(a) shows a wide-field en-face SECM image with the corresponding H&E histology in Fig. 2(b) for a rat lung slice approximately 70 μm in depth from the top surface of the slice. The wide-field SECM image demonstrates the ability to visualize relatively large fields of view with cellular-level detail. Figure 2(c) to 2(d) shows how a zoomed-in region of a SECM image with corresponding histology to demonstrate how the airway epithelium, vessel endothelium and blood, and aggregations of cell nuclei can be characterized by bright SECM signal. Type II alveolar pneumocytes (arrows) were directly matched between SECM and histology.

Fig. 2

SECM and H&E histology images of PBS-immersed fixed rat lung slice. (a) En-face SECM image 70 μm within tissue and (b) corresponding H&E histology, where V indicates a vessel and $\mathrm{scale}\mathrm{bar}=500\mathit{\mu }\mathrm{m}$. (c) SECM image in greater detail and (d) corresponding H&E histology, where arrows indicate alveolar type II pneumocytes and $\mathrm{scale}\mathrm{bar}=100\mathrm{\mu m}$.

The maximum imaging depth was dependent on the structure. Blood filled vessels and the surrounding collagen matrix provide high contrast up to 150 μm into the specimen, but alveolar walls were difficult to identify for imaging depths greater than 100 μm. Thirty randomly chosen alveolar walls were measured with a FWHM thickness of $5.5\pm 6.1\mathit{\mu }\mathrm{m}$ ($\text{mean}\pm \text{standard}\text{deviation}$). It is important to note, that in some histology slides, the structures appear to be distorted or reduced in size, which may have been caused by histologic tissue processing required to obtain the H&E stained slides. Although best efforts were made to ensure that the SECM and H&E section were parallel, there is likely some tilt in the H&E image versus the SECM image due to the embedding and sectioning process.

3.3.

FFOCM

Figure 3(a)–3(c) shows images obtained from a rat lung slice with FFOCM from all three spatial planes, with H&E histology from the same sample in Fig. 3(d). The alveolar walls can be clearly delineated and the capillary network inside the walls was visible as non-scattering media surrounded by the scattering endothelium (arrows). Cells that are likely to be type II alveolar pneumocytes (stars) are characterized as highly scattering with a relatively large size and shape compared to what would be expected for type I pneumocytes. However, individual type I pneumocytes could not be clearly identified. Blood vessels (V) were identified and their endothelium was distinguished from the blood content due to high scattering from the nuclei of the endothelial cells. Blood cells could also be visualized with a crenated appearance, likely due to the fixation process. The maximum imaging depth reached beyond 250 μm when imaging through the PBS-immersed airspaces, but the strong blood attenuation limited the imaging depth to less than 50 μm when imaging through blood filled vessels. Thirty randomly chosen alveolar walls were measured with a FWHM thickness of $4.8\pm 3.3\mathit{\mu }\mathrm{m}$ ($\text{mean}\pm \text{standard}\text{deviation}$).

Fig. 3

FFOCM and H&E histology images of PBS-immersed fixed rat lung slice. (a) En-face, (b) axial cross-section, and (c) orthogonal axial cross-section images obtained with FFOCM, where dotted lines show the orientation of axial cross-sections intersecting at a three-dimensional type II pneumocyte. (d) Representative H&E histology slide from nearby location sectioned in en-face plane. Stars indicate alveolar type II pneumocytes, V indicates a blood vessel containing crenated blood cells, and dotted arrows indicate capillaries in alveolar walls. $\mathrm{Scale}\mathrm{bars}=100\mathit{\mu }\mathrm{m}$.

3.4.

Comparison of Imaging Techniques

Figure 4 compares images from all three reflectance imaging techniques of a fixed PBS-immersed rat lung slice acquired within the same field of view. Figure 4(a)–4(c) shows en-face OFDI and SECM sections with the corresponding histology; and Fig. 4(d)–4(f) shows zoomed-in regions of the OFDI and SECM images that correspond to the FFOCM field of view. These images illustrate the difference in resolution and expected image quality between the techniques. Table 2 summarizes the performance of OFDI, SECM, and FFOCM for imaging PBS-immersed, fixed rat lung tissue slices, as described below.

Fig. 4

OFDI, SECM, FFOCM and H&E histology images of PBS immersed fixed rat lung slice. (a) OFDI, (b) SECM, and (c) H&E histology within the en-face plane, where $\mathrm{scale}\mathrm{bar}=500\mathit{\mu }\mathrm{m}$. (d) OFDI and (e) SECM images in greater detail matched with (f) FFOCM image, where $\mathrm{scale}\mathrm{bar}=50\mathit{\mu }\mathrm{m}$.

Table 2

Summary of performance criteria for current OFDI, SECM, and FFOCM systems to image PBS-immersed rat lung tissue.

OFDI provided high frame rates and the largest imaging depth for the specific systems used in this study. The lower transverse resolution of the system resulted in images with thicker alveolar walls, but the majority of the airspaces and vessels were still visualized within the rat lung samples. OFDI might therefore provide the most practical option for evaluating alveoli over a relatively large tissue volume. The high acquisition speed may also enable alveolar imaging where motion artifacts hinder current technologies such as micro-CT.

OCT imaging has already been performed during bronchoscopy and visualized intact alveolar attachments to the small airways.²⁶ As catheters become smaller, more regions surrounding thin walled airways could be reached in a minimally invasive way that limits any changes to the alveolar environment. Currently, 0.8 mm diameter optical catheters are used in the clinical setting to assess coronary arteries.²⁷ It is important to note that bronchoscopic access limits the analysis to alveolar features immediately surrounding the airways.

Subpleural alveoli could be evaluated in an open-chest procedure or through an optical window as previously demonstrated in a rabbit model by Meissner et al.⁶ In addition to requiring mechanical ventilation, this route of access currently removes any local effect the chest wall has on the alveolar environment. Lastly, OCT-based needle probes have also been demonstrated^19,28,29 to provide access to lung parenchyma that cannot be visualized through the airways or the pleura with optical imaging techniques. However, this route of access comes at the cost of local trauma to the tissue and the possibility of pneumothorax as well as drag artifacts caused by needle movement.

SECM provided much higher resolutions in the lateral (1.3 μm), and axial direction (2.4 μm) than OFDI and could visualize type II pneumocytes. The high resolution of SECM could also provide absolute measurements of alveolar wall thickness and identification of inflammatory cells. The usable imaging depth with the current system configuration was 100 to 150 μm, which was the lowest of the three techniques. The potential of SECM for imaging at high speeds may facilitate the acquisition of large fields of view and dynamic imaging, but miniature probes are not currently available. Forward-scanning and helical-scanning probes have been demonstrated at diameters of 10 mm^23,30,31 with the promise to further reduce the probe diameter up to 5 mm.³¹ At this development stage, only imaging through a pleural window or an open chest procedure seems possible.³² To enable endoscopic imaging, probe diameters $\le 1\text{mm}$ would be most practical, allowing the probe to be inserted into the working channel of any adult bronchoscope and reach later generations with thinner airway walls. Confocal fluorescence microscopy has been demonstrated for in vivo alveolar imaging through a bronchoscope and via penetration through the distal bronchiolar wall.³³ A confocal imaging probe to be inserted in a 22 gauge needle has recently been developed for biological tissue.³⁴

FFOCM provided the best spatial resolution of all three techniques and would be best suited to evaluate absolute alveolar wall thickness in different species and pathophysiologic changes such as wall thickening or inflammatory cell recruitment. The 1-μm isotropic resolution of FFOCM provides the ability to analyze 3-D structures down to a few micrometers, which could potentially be useful for identifying and characterizing individual cells such as alveolar type I or II, macrophages or eosinophils. FFOCM currently provides the slowest acquisition speed of the three imaging systems, however, and the development of miniaturized probes for both in vivo and ex-vivo imaging is still at an early stage. A FFOCM system with 1.5 million  A-lines/s acquisition has been demonstrated with a spatial resolution around 13 μm.³⁵ A miniaturized 2 mm diameter probe has also been demonstrated with a resolution of 3.5 and 1.8 μm in the lateral and axial direction, respectively.³⁶

3.5.

Study Limitations

This study investigated the use of three reflectance optical imaging techniques to image lung tissue from a single species, the rat. However, we expect our conclusions to translate to all mammalian species, including the ability to visualize cells (where applicable), airspaces, blood vessels, and alveolar walls within the specified imaging depths. The structures of the alveolar regions of the lung are conserved across all mammalian species, with only a change in alveolar size and alveolar interstitial thickness.³⁷

In this paper, we investigated the advantages and limitations of the different technologies with respect to resolution, field of view, and imaging depth. These parameters are in part specific to the current device configurations. For example, the OFDI configuration implemented within this study could not resolve alveoli less than 20 to 30 μm in diameter, which could be limiting in a mouse model. However, this limitation is not intrinsic to the technology as high-transverse resolution OFDI can be performed with a higher NA lens. It is reasonable to expect improved cellular detail within a decreased imaging depth by implementing a larger NA lens within the OFDI sample arm probe. SECM, as implemented here, obtained images using a swept-source laser with a 5 kHz line rate, resulting in several minute acquisition times. Newer lasers with rates up to and exceeding 1 MHz have been developed, and it is likely the combination with high speed detection systems would allow imaging at image acquisition speeds that are 1 to 2 orders of magnitude greater than video rate. It is also important to note that the field of view of the current FFOCM system might present difficulties when attempting to image multiple alveoli within larger species. Again, this limitation is specific to the imaging system we used in the study and not intrinsic to the technique itself.

All samples were filled with PBS and not air, as would be the case in a normal respiring lung. Although some alveoli could in principle be fluid filled during various clinical conditions, such as bronchoalveolar lavage, liquid breathing, or edema, additional challenges will arise when translating these technologies for imaging air-filled alveoli in vivo. It is important to note that significant differences in image quality are expected for fluid- versus air-filled alveoli as light refracts substantially when encountering high refractive index differences between tissue and air. We therefore expect the imaging depth of the tested technologies to be greatly reduced when the alveoli are filled with air, as previously demonstrated in ethanol-filled rabbit lungs using spectral-domain OCT,³⁸ that resulted in distorted alveolar shapes.

This study was conducted in fixed lung tissue for practical reasons. In doing so, the same field of view could be imaged with all three technologies at the same inflation state. Furthermore, the lung could be cut into slices, which allowed the evaluation of any location within the lung compared to limiting the analysis to the alveolar layers directly beneath the pleura. The difference in refractive index between fresh and fixed tissue impacts both the resolution and imaging depth. Fixed tissue has a higher refractive index due to the polyethylene glycol (1.47 at 20ºC) and we expect PBS-immersed fresh lungs will show decreased refraction artifacts and slightly increased imaging depth at the cost of a decrease in contrast and/or resolution. In addition, light scattering by unfixed blood will decrease the imaging depth depending on the size of the vessel within the field of view.³⁹

We have presented a limited number of direct correlations to histology, which does not allow verification of all structures visible in the images. Due to the additional processing cycles between image acquisition and H&E sectioning, the fine structures of the alveolar walls are distorted and difficult to recognize. Often intact histology sections could only be obtained after cutting up to 100 μm from the tissue surface. Correlative histology was also difficult to find due to the limited imaging depth of the technologies. While these difficulties limited our ability to analyze the data, they emphasized the importance of imaging techniques that provide non-destructive assessment of alveolar structure and function.