|
|
||||||||
J Thorac Cardiovasc Surg 2005;129:615-622
© 2005 The American Association for Thoracic Surgery
Evolving Technology |
a Beckman Laser Institute, University of California, Irvine, Irvine, Calif
b Pulmonary and Critical Care Division
d Cardiothoracic Surgery, UC Irvine Medical Center, Orange, Calif
c Veterans Administration Medical Center, Long Beach, Calif
Read at the Thirtieth Annual Meeting of The Western Thoracic Surgical Association, Maui, Hawaii, June 23-26, 2004.
Received for publication June 22, 2004; revisions received October 7, 2004; accepted for publication October 15, 2004. 1 Address for reprints: Matt Brenner, MD, Pulmonary and Critical Care Division, UC Irvine Medical Center, Bldg 53, Rm 119, 101 City Drive South, Orange, CA 92868 (E-mail: mbrenner{at}uci.edu).
2 Zhongping Chen, PhD, Department of Biomedical Engineering, Beckman Laser Institute, UC Irvine, Irvine, CA 9261 (E-mail: Zchen{at}bli.uci.edu).
| Abstract |
|---|
|
|
|---|
METHODS: Freshly excised lungs and pleural tissue obtained from rabbits with inhalation lung injury and induced empyema, metastatic sarcomas, and pleural sarcomas and from patients with airway disease were imaged by using 2- and 3-dimensional optical coherence tomography with a prototype superluminescent diode optical coherence tomographic system constructed in our laboratory. Lungs and pleural tissue were subsequently processed for standard hematoxylin and eosin histology for comparison with optical coherence tomography.
RESULTS: Optical coherence tomographic imaging achieved an ex vivo resolution of 10 µm and an in vivo resolution of about 30 µm with a depth penetration of 1 to 2 mm with 2- and 3- dimensional reconstruction capabilities. Tumors as small as 500 µm were detectable with optical coherence tomography. The acquired images closely matched histologic images, demonstrating details at the level of mucosal layers, glands, alveoli, and respiratory bronchioles.
CONCLUSIONS: Optical coherence tomography with near-infrared interferometric methods enables near real-time in vivo near-histologic resolution optical imaging. With further advances, optical coherence tomography has the potential for real-time accurate and early pleural and subpleural diagnostics by using small-diameter flexible fiberoptic endoscopic probes for a wide range of thoracic surgical applications.
|
During bronchoscopy or endoscopy or while in the operating room, the ability to distinguish benign from malignant disease and to determine the extent of lesion margins is critical for successful interventions. In addition, rapid and accurate evaluation of airway pathologic changes is important for minimizing morbidity and mortality in other conditions, such as acute burn or inhalation injury.1,2 Until now, assessment for proper clinical management relied primarily on visualization of abnormalities during endoscopic biopsy on frozen sections sent to pathology and gross inspection at the time of surgical intervention. A means to obtain real-time noninvasive histologic imaging would aid in diagnosis, help to ensure higher-yield biopsy samples, potentially save considerable operating time, and possibly help avoid unnecessary interventions or repeated procedures.
Optical coherence tomography (OCT) is emerging as a rapid-acquisition, high-resolution imaging modality that provides capabilities for real-time near-histologic evaluation.3 In attempting to approach the concept of optical biopsy,4 OCT offers the potential for surface and subsurface optical imaging (up to a depth of 1-2 mm) with high spatial resolution of tissue microstructure without requiring contact between the optical probe and the tissue sample.3 Tissue layers, glands, small blood vessels, and cartilage can be visualized at theoretic resolutions approaching 10 µm with the use of superluminescent diode (SLD) laser prototype systems.5 This technology, in combination with minimally invasive techniques, can be applied in the surgical field to examine tissue microstructure, guide biopsies, and minimize the need for frozen sections or the uncertainties associated with gross examination.6 Additionally, multiple sequential high-resolution images can be captured, rendered, and integrated to form 3-dimensional images.7
We developed a fiberoptic OCT probe (1 mm in diameter) for bronchoscopic and thoracoscopic application to assess the feasibility of OCT imaging in inflammatory and neoplastic changes of the airway and lung parenchyma, as well as of visceral and parietal pleural tumors, induced by a novel pleural malignancy animal model developed in our laboratory. Two- and 3-dimensional OCT images were constructed. This technology was then applied to patients with known or suspected airway malignancy.
| Materials and methods |
|---|
|
|
|---|
Airway injury and empyema induction
Twelve animals were inoculated with 1.9 x 103 to 2.4 x 105 Streptococcus pneumoniae cells by using a sterile pediatric suction catheter in accordance with an approved protocol from the Institute of Surgical Research, San Antonio, Tex.8 Animals were monitored on the basis of vital signs, blood work, and pulmonary function tests at the time of and after 24, 48, 72, and 96 hours of exposure. Computed tomographic scans, flow cytometry, and bronchoalveolar lavage fluid culture were performed to confirm the diagnosis of pneumonia. On the fourth day after inoculation, surviving rabbits were sacrificed, and their tracheas were excised, placed in isotonic saline, packed on ice, and sent overnight to the Beckman Laser Institute. All samples were imaged within 3 days of excision. Empyemas were induced in a New Zealand White rabbit, as previously described.9 In brief, 2 mL of 108 Pasteurella multocida bacteria (in 0.5% Brain Heart Infusion agar) was injected into the right pleural space. Twenty-four hours after induction, pleural fluid from a diagnostic thoracentesis was cultured and analyzed for pH and glucose to verify the presence of empyema. After day 6 of induction and after the animals were sacrificed, the thorax was dissected en bloc, and specimens from the right middle lobe with attached visceral pleura were collected. These specimens were frozen at 20°C until the time of OCT imaging.
Pleural and parenchymal tumor implantation
After intubation, a 4-mm thoracoscope (Karl Storz, Tuttlingen, Germany) was inserted under sterile conditions in the fifth or sixth intercostal spaces of 12 New Zealand White rabbits, and with thoracoscopic visualization, a small section of the chest wall parietal pleura was mildly abraded with the wooden end of a sterile cotton-tip applicator. A syringe containing a dose of 10 to 13 million VX2 sarcoma tumor cells (M.D. Anderson Cancer Center, University of Texas, Houston, Tex) excised and prepared for suspension from rabbits with primary tumors was used to inject the cells into the chest cavity adjacent to the abraded pleural surface.
Hematogenous tumor implantation
Hematogenous lung metastases were induced either by means of direct intravenous injection of the tumor cells into the rabbit ear vein or by means of intramuscular injection into the left thigh (10-15 million cells) in 12 New Zealand White rabbits. Hematogenous tumor spread to the lungs generally occurred 2 to 4 weeks after intravenous or intramuscular injection.
OCT and flexible fiberoptic probe prototype
OCT theory has been discussed in detail in previous studies.3 in brief, OCT uses a broadband near-infrared light source in which the emitted light is split into sample and reference beams. The reflected waveforms combine to create an interference pattern (Figure E1, a). A low temporal coherence SLD light source (central wavelength
o = 1300 nm and full width at half maximum = 
= 80 nm; AFC Technologies, Hull, Quebec, Canada) is connected to a Michelson interferometer that splits the light source into a sample and reference beam. These reflected beams recombine at the fiber coupler in the interferometer, producing the interference pattern detected by a photomultiplier.
|
= 650 nm) was coupled to the system to elucidate the location of the sampling site. Flexible fiberoptic OCT probes (Figure E1, b) were constructed from single-mode fiber patch cord (ThorLabs, Newton, NJ). The bare-ended fiber was attached to a 0.5-mm-diameter GRIN lens (NSG America, Irvine, Calif) with optical adhesive (Optical Adhesive no. 68; Norland Products, Cranbury, NJ) under a microscope. A right-angle light path was achieved by using a 0.5-mm prism.10 The probe was placed in FEP tubing (17-gauge thin wall; Zeus, Orangeburg, SC) for added fiber support.
Tracheal and lung OCT
Tracheal OCT
After anesthesia induction and intubation, the trachea was exposed by means of blunt dissection so that localization of OCT sampling could be determined. A flexible fiberoptic probe was inserted into the endotracheal tube. The visible laser light emitted from the probe confirmed the segment of trachea under OCT investigation, and OCT images were obtained. After animal death, the trachea was removed. Twelve excised tracheas were cut open longitudinally along their musculofibrous membranes and divided into approximately 29 sections of 2 cm in length. Triangular notches were cut into the opposite ends of each specimen to delineate the line of image acquisition perpendicular to the cartilage rings (Figure 1, b). The tracheas were secured to pieces of cork by using metal pins placed along their perimeter and covered by a layer of KY jelly to prevent desiccation during imaging. The tracheas were placed on a moveable sample platform, and a visible-light guiding beam was used to match the line of image acquisition with the triangular marking notches.
|
Two- and 3-dimensional OCT
OCT images were performed on 8 excised tissues from rabbits and purchased pig lungs during the probe developmental stage in a similar fashion to images performed during in vivo studies. Specimens were mounted on cork board, and by using a 4x objective attached to a servo motor, the coherent laser light source was focused through the lens and scanned over the tissue. The specimen was advanced along the x-axis 20 or 40 µm (depending on desired resolution) after each single lateral pass on the y-axis to accomplish 3-dimensional OCT. Three-dimensional images of OCT and histology sections were constructed with the aid of a software package (3D Doctor; Able Software Corp, Lexington, Mass). The time to scan a 3-mm3 (1 x 3 mm) tissue was approximately 20 minutes.
Human airway OCT imaging
During clinical bronchoscopies in patients with known or suspected airway disease, the flexible fiberoptic probe was inserted through the working channel of the bronchoscope. The suspicious region was imaged with OCT. If clinically indicated, a biopsy was performed at the imaged site and prepared in the standard manner for histologic analysis. Human studies were approved by the institutional review board (no. 2003-2907). Informed consent was obtained before bronchoscopic procedures.
Histology
Histology of excised tissue was prepared according to standard hematoxylin and eosin (H&E) histologic staining methods. OCT images and those of the histologic sections were compared. Tissue slide examination and micrographs were performed with an Olympus BH2 light microscope (Olympus American, Melville, NY) and recorded with an Olympus DP10 camera (Olympus American) for a light microscope and Olympus Digital Microfire 1.0 (Olympus American).
| Results |
|---|
|
|
|---|
Although there was variability within samples from each image, marked differences between the normal lungs and pleura compared with the malignant samples were clearly visualized with OCT. Changes ranged from loss of structure to variations in tissue thickness.
Tracheal OCT
In vivo endoscopic OCT images displayed tracheal structures visible even through the endotracheal tube (Figure 1, a). Clear anatomy of mucosal, submucosal, and cartilage layers are seen.
OCT images and the corresponding histology sections are shown in Figure 1, c and d, and Figure 1, e and f, for both normal and inflamed trachea, respectively. Tracheal cartilage (Figure 1, c) was an identifiable landmark seen during OCT imaging, which was confirmed with histology. OCT images were able to distinguish the submucosa from the lamina propria. Also observed in both OCT mapping and histology sections were submucosal glands between cartilage rings. A slight variation in tissue structure occurred, with decreased submucosal layer thickness in the histologic specimens, which was believed to have resulted from tissue property desiccation changes after excision and fixation. Injured trachea obtained from animals inoculated with S pneumoniae demonstrated a thickened submucosa that was seen in both OCT imaging and histology (Figure 1, e and f).
Lung and pleural OCT
OCT imaging of lung tissue was able to detect structures as fine as alveolar septa and visceral pleura. Figure 2, a and b, and Figure 2, c and d, show comparisons between OCT and histology of normal pig and rabbit lung specimens, respectively. In Figure 2 alveoli and visceral pleura were discernible. OCT images of pig lung are superior to those of rabbit lung, possibly because of the finer structures in the rabbit lung. However, in the rabbit lung tissue with induced bacterial empyema (Figure 2, e) and metastatic disease (Figure 2, g and h), these structures were no longer distinguishable because of thickening of the pleura and filling of the alveoli with purulent material (Figure 2, f) and the compression of adjacent tissues by lung tumor Figure 3.
|
|
|
Three-dimensional imaging of OCT
In selected specimens 3-dimensional reconstructions were prepared and analyzed for comparison with 3-dimensional histology. Although some differences caused by fixation of the histologic slide tissues were noted, both OCT and histologic 3-dimensional images demonstrated very similar appearance. Figure 4, g through j, shows 3-dimensional relationships between OCT images and histologic preparations.
Human airway
Two patients with and without airway disease who provided informed consent (protocol HS#2003-2907) underwent clinical flexible fiberoptic bronchoscopies with OCT. The OCT probe was passed through the working port of the flexible fiberoptic bronchoscope, and OCT images were obtained of the intrabronchial lesions. Lateral scanning sampling resolution averaged 20 to 30 µm in the lung. Images showed changes in airway structure in malignant airway regions when compared with normal tissue. Pathologic changes noted on OCT consisted of a thickened mucosa and disorganization of tracheal tissue layers when compared with sections of normal tissue. Corresponding OCT and white-light bronchoscope images of inflamed tissues obtained during bronchoscopy are shown in Figure 5.
|
| Discussion |
|---|
|
|
|---|
Histologic examination of bronchial mucosa during flexible or rigid bronchoscopic procedures could greatly assist in the diagnosis and localization of endobronchial malignancy. This might be particularly useful in malignancies such as adenoid cystic carcinomas in which tumor spread tends to occur in the submucosal plane well beyond the observed luminal component of the tumor. Application of OCT in this and similar settings would allow for possible detection of hidden margins (Figure 2, g) that might otherwise be missed in gross examination and could assist with guiding resection margins, as well as assessing operability.
Tissue structural changes strongly suggestive of malignant tumor invasion could be detected at resolution of the current SLD OCT system (Figure 4). However, improvement in resolution to 1 to 2 µm will be required to visualize nuclei and organelles to definitively differentiate malignant transformation optically at a cellular level. Such improvements in resolution might be achievable in the near future by using broadband light sources, polarization-sensitive OCT (PS-OCT), and multiphoton microscopy combined with OCT.18
The ability to visualize tissue structures in real time at a near-histologic level of resolution opens up a wide range of potential areas for clinical and research applications in thoracic surgery. In the future, when cellular-level OCT resolutions are obtained, even greater uses for OCT in thoracic diagnostics can be envisioned. However, the depth of penetration of OCT is relatively shallow (1-2 mm) and is likely to remain limited by the degree of scattering inherent in complex biologic tissues of the lung and thorax, and thus major advances in depth of penetration are unlikely. Nevertheless, there are many scenarios in thoracic surgery in which surface and near-surface, in vivo, high-resolution optical imaging are of great potential value.
The development of 3-dimensional high-resolution OCT probes, endoscopes, and image-processing and display technologies might allow for improved assessment of tumor margins and detection of satellite lesions. Further into the future, very small OCT probes could be designed to fit within needles to allow imaging at depths within masses with near real-time histologic resolution capabilities.
This study has demonstrated the feasibility of high-resolution 2- and 3-dimensional OCT for examination of thoracic airway and pleural abnormalities. There are a range of potential research and clinical applications for OCT imaging in thoracic surgery and diagnostics. The technology used in this study is limited to 10- to 30-µm resolutions, and depth of penetration is generally a maximum of 2 to 3 mm. With further improvement in resolution, contrast, acquisition, display, and processing and the development of specific thoracic probes, OCT might offer a significant advance for the diagnosis and treatment of patients with thoracic diseases.
| Acknowledgments |
|---|
| Footnotes |
|---|
| References |
|---|
|
|
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |