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Endobronchial, OCT or OFDI is performed using thin flexible catheters, which are compatible with standard bronchoscopic access ports. The bronchoscope is advanced within the tracheal bronchial tree to the desired airway. The OFDI catheter is then advanced into the airway through the bronchoscope working channel to collect images.

The inner optical core of the OFDI catheter is rotated and simultaneously pulled back within the outer transparent sheath to generate three dimensional OFDI Data. A needle based OFDI catheter has been developed by the Sudor Laboratory at Massachusetts General Hospital, which is compatible with standard transbronchial needles routinely used to sample peripheral masses. The bronchoscope is advanced within the airway to the targeted mass.

With the OFDI catheter safely retracted into the transbronchial needle, the needle is inserted into the mass. The needle is subsequently retracted, leaving the OFDI catheter in place. As with Endobronchial, OFDI, the inner optical core of the Transbronchial OFDI catheter is rotated and simultaneously pulled back within the outer transparent sheath to generate three dimensional OFDI.

Data histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI data sets. As a result, specific imaging features of pulmonary pathology cannot be easily developed in the in vivo setting. The overall goal of this protocol is to obtain precisely matched one-to-one OFDI and histology correlations with ex vivo tissue, which is vital to accurately evaluating features seen in OFDI against histology as a gold standard.

Once specific imaging criteria has been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. The main advantage of this technique is it allows for direct correlation between imaging and histology, which enhances image feature interpretation. To prepare the optical frequency domain imaging or OFDI system for the experiment, first turn the imaging system on and then set and record appropriate imaging parameters.

Attach the catheter to the rotary junction and pullback device. This custom built catheter has a diameter of 0.8 millimeters. Spin the catheter and check for image quality image, a gloved fingertip to ensure there is an image that is in focus and the signal to noise ratio is adequate.

To begin the procedure for tissue preparation, place a tabletop disposable absorbent pad on the benchtop and set the lung specimen on the pad. Due to scheduling difficulties in obtaining tissue from patients, swine tissue will be used. In this video demonstration.

Identify the bronchial airway entering the resection specimen at the hilum. Using a syringe bore, remove any visible mucus from within the airway. Palpate the exterior surface of the specimen to identify an airway or the lesion of interest.

Open the airway along the probe until the lesion of interest is visible or palpable under the airway mucosa with a cotton tipped applicator. Carefully remove any blood or mucus from the airway mucosa overlying the lesion. Place the OFDI catheter above the airway mucosa and obtain an image.

This is to confirm that the lesion is underlying the airway mucosa and to identify a high quality imaging region of interest for histology correlation. After selecting the region of interest in the airway, choose two points on the tissue along the desired line of imaging. In this example, the points will be made parallel to the longitudinal aspect of the airway.

Making the ink marks visible on both OFDI and histology can be challenging, which is why we use both needle marks and ink in order to see the marks in both techniques. Once the two points are chosen, dip a fine tipped open bore needle into the tissue marking dye carefully wipe excess ink off the outside of the needle with gauze leaving tissue marking ink only within the needle bore puncture the tissue perpendicular to the airway mucosa at the chosen point along the line of imaging. In the same way, mark the second point on the airway mucosa, making sure the second point is no more than 1.5 centimeters away from the first point.

If the ink runs over the mucosal surface away from the puncture site, use a cotton tipped applicator to carefully remove the excess ink. Depending on the desired results, the two points may also be made parallel to the circumferential aspect of the airway, as shown here. To image the tissue, place the OFDI catheter over each ink mark and image to ensure the marks are visible on OFDI.

Marks should appear as focal disruptions within the tissue structure with overlying highly scattering particles and underlying rapid signal attenuation, which corresponds to the ink particles within the puncture site. In this OFDI image of swine airway, both ink marks are indicated by asterisk. Place the catheter parallel to the two ink marks on the airway mucosal surface, such that the catheter optics overly the tissue beyond the first ink mark.

Proceed with collecting an OFDI pullback view the OFDI pullback images to ensure both ink marks are visible in imaging and to check for motion artifacts prior to collecting the tissue. Place a green ink dot on the airway mucosal tissue 0.3 centimeters away from the ink mark that appeared first in the imaging pullback. This is to orient the beginning of the imaging scan.

Now remove the tissue encompassing the two black ink marks and green ink mark. Trim the tissue to fit into a standard histology processing cassette. Place the tissue in a histology processing cassette and fix in 10%formalin for at least 48 hours.

Subsequently, the tissue is processed as described in the protocol text. A representative result of OFDI is illustrated by these in vivo images obtained from swine airway Under mechanical ventilation. Panel A is an OFDI cross section of the proximal airway, and panel B is an OFDI cross section of the distal airway.

Panel E is an OFDI longitudinal section of the airway from proximal on the left to distal on the right panel C is a higher magnification of the red highlighted region from panel E.Panel D is a higher magnification image of the green highlighted region from panel E.The catheter diameter is 0.8 millimeters and the tick marks represent 0.5 millimeter increments. Although different layers of the airway wall and alveolar attachments are discernible in the OFDI images, it is difficult to precisely interpret the anatomic correlate of the OFDI signals without directly registered histology. E indicates the epithelium lp, the lamina propria SM the submucosa, C, the cartilage, and a the alveolar attachments.

Using the protocol demonstrated here a precise correlation between OFDI and histology is achieved. This OFDI image of swine airway shows precise correlation with histology of tissue sections stained with H and E orme. The asterisks indicate the black ink marks visible on the respiratory epithelium.

Airway layering can be visualized on the H and e and tri RME stains. A higher magnification view of the OFDI image is shown here with corresponding histology stained with h and e and tri rme E indicates the respiratory epithelium el the dense collagen and elastic tissues SM the smooth muscle C.The cartilage rings G, the salivary gland tissue, and D, the salivary duct entering the epithelium. Smooth muscle appears as discontinuous interspersed fales and is thus not identifiable in OFDI.

Similarly, using tissue marking a precise correlation between OFDI and histology is demonstrated in the human airway. In this OFDI image of the human proximal airway, both ink marks are visible and indicated by asterisk. There is precise correlation with histology of tissue sections stained with h and e and tri chrome in the H and e stain.

The black ink marks are visible on the respiratory epithelium indicated by asterisk higher magnification views of the OFDI image and corresponding histology. Stained with h and e and tri CHRO are shown in the inset images. E indicates the respiratory epithelium lp, the Lamin propria G, the salivary gland tissue C, the cartilage rings, and pc, the perichondrium in the human airway.

Typical layering is visible. Interspersed within the loose connective tissue are fales of red staining smooth muscle shown in panel C and F, which do not form a continuous band and are thus not visible as a distinct layer in OFDI. These two images show the results of OFDI of human distal airway and the precisely correlated h and d histology.

The black ink marks visible on the respiratory epithelium are indicated by asterisk alveolar attachments indicated by a are visible as signal intense lattice like alveolar walls with signal void alveolar spaces. Vascular spaces within the lamina propria are also visible as signal void structures with underlying mild shadowing indicated by arrows. E is the epithelium and LP is the lamina propria.

The rapid acquisition rates inherent in OFDI allow for large area imaging. The data can be viewed in both the cross-sectional and longitudinal aspects to provide additional architectural information about the tissue. Once imaging features have been developed with this technique, the imaging features may be used to interpret imaging regions which do not have matched histology.

This technique provides a method to interpret imaging features with directly registered histology. Although this technique was demonstrated using OFDI and lung tissue, it can be applied to many imaging techniques and tissue types. Once imaging features of normal and disease tissue have been established with this technique, the imaging features can then be applied to in vivo image interpretation, where directly correlated histology may not be available.