Objective
The objective of the study was to develop a clinical confocal microlaparoscope for imaging ovary epithelium in vivo with the long-term objective of diagnosing cancer in vivo.
Study Design
A confocal microlaparoscope was developed and used to image the ovaries of 21 patients in vivo using fluorescein sodium and acridine orange as the fluorescent contrast agents.
Results
The device was tested in vivo and demonstrated to be safe and function as designed. Real-time cellular visualization of ovary epithelium was demonstrated.
Conclusion
The confocal microlaparoscope represents a new type of in vivo imaging device. With its ability to image cellular details in real time, it has the potential to aid in the early diagnosis of cancer. Initially the device may be used to locate unusual regions for guided biopsies. In the long term, the device may be able to supplant traditional biopsies and allow the surgeon to identify early-stage ovarian cancer.
In this article, we present the first known use of a confocal microlaparoscope in humans. This new type of laparoscope allows surgeons to obtain nondestructive optical biopsies of tissue in real time. We present results from a pilot study consisting of 21 patients. The device was used to image ovaries in vivo prior to laparoscopic oophorectomy.
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The American Cancer Society estimates that more than 230,000 new cases of ovarian cancer were diagnosed in 2007, and more than 141,000 women died from the disease worldwide. More than 80% of all cases are diagnosed during the later stages (stage III or IV) of the disease, when treatment is expensive and generally unsuccessful. If diagnosis occurs when the cancer is localized to the ovary (stage I), the survival rate increases to 92%. The overall prevalence of ovarian cancer is relatively low (1.7% lifetime risk). However, the lifetime risk for the disease increases 2-fold if the individual has 1 first- or second-degree relative with the disease. With 2 first-degree relatives, the risk increases by 25 times (a 40% lifetime risk).
For the subgroups of women at increased risk, few options exist to allow early detection of the disease. A National Institutes of Health 1994 consensus stated that there is no single acceptable screening test for ovarian cancer and no evidence that combining the available screening tests, CA-125, transvaginal ultrasound, and pelvic examination, has an acceptable sensitivity and specificity.
Several novel techniques are being developed that could provide clinicians with diagnostic tools to detect early changes in ovarian tissue. Techniques such as confocal microscopy, 2 photon microscopy, optical spectroscopy, and optical coherence tomography are able to assess tissue morphology and/or biochemical composition. In vivo use of these methods has the potential to provide real-time diagnostic information, allowing clinicians to assess the subtle changes that occur early in the disease process. These techniques may increase the sensitivity and specificity of a diagnosis, especially when used as a means for targeting traditional biopsies to regions that can be identified as abnormal at the cellular level.
Confocal microendoscopy, an emerging in vivo fluorescence imaging technique, can resolve individual cells and subcellular features. The confocal microendoscope is able to image thin sections of thick samples. This optical sectioning property enables high-resolution microscopic imaging of thick biologic samples at moderate depths (typically up to 100–200 μm below the tissue surface).
In vivo confocal imaging has the potential to aid clinicians during screening and/or surgical procedures. Commercial confocal microendoscope systems have recently been developed for visualization of colon and esophagus. Results have been encouraging in these areas, and additional applications for this technology are under investigation. The purpose of this article is to describe the first clinical confocal microlaparoscope system. In the following sections, we describe the new device and present the results of a pilot study.
Materials and Methods
We previously reported on the development of a multispectral fluorescence confocal microendoscope. To evaluate whether such a device could be used to image ovaries in vivo, a clinical confocal microlaparoscope system was developed. To create a viable clinical system, several criteria had to be met. First, the microlaparoscope system had to be compact and mobile so that it could be quickly moved into the surgical suite. The microlaparoscope also had to be compatible with standard trocars, be sterilizable, have the ability to deliver small localized volumes of contrast agents, and have the ability to focus at selected depths. Finally, the device had to be comfortable and easy to use during surgery. A confocal microlaparoscope system was constructed meeting these criteria.
To view real-time cellular images using the confocal microlaparoscope, the surgeon places the rigid probe through a 5-mm trocar port and contacts the epithelial surface of the organ. Pressing a button on the microlaparoscope handle delivers a controlled volume of fluorescent contrast agent to the field of view. Instantly, a live video of the cells appears on screen. As the probe tip is moved across the surface of the organ, the epithelium can be interrogated in real time. Pressing another button on the handle saves videos and still frames. By default, the system is configured to image the epithelial layer of cells. However, controls located on the handle allow the surgeon to view deeper cell layers. After use, the microlaparoscope can be disconnected from the system, cleaned, and ethylene oxide (ETO) sterilized for reuse.
In the following paragraphs, we describe the confocal microlaparoscope system, discuss the fluorescent contrast agents that were used during imaging, and describe the imaging protocol used to test the device.
Confocal microlaparoscope system
The confocal microlaparoscope system consists of a microlaparoscope connected to a mobile cart containing a confocal optical scan unit. The microlaparoscope ( Figure 1 ) has a 35-cm rigid probe extending from its handle. The handle contains controls that allow the surgeon to adjust the focus, deliver contrast agents, save still images, and record videos. A flexible cable connects the microlaparoscope to the mobile cart.
The tip of the microlaparoscope’s probe contains a miniature 3-mm diameter objective lens that provides a 450-μm field of view and a 3-μm lateral resolution. The miniature objective lens images the tissue plane onto a flexible coherent fiber-optic imaging bundle (Sumitomo Electric Industries, White Plains, NY). The fiber bundle runs through a 6-m long cable that connects to the optical scan unit, located on the mobile cart. To adjust the imaging depth in the tissue, the focus motor inside the microlaparoscope’s handle changes the spacing between the fiber bundle and the objective lens.
To deliver contrast agent to the imaging site, a syringe with fluorescent dye is placed into a spring-loaded port in the handle of the device. The second motor in the microlaparoscope handle acts as a syringe pump that forces the contrast agent through a tiny fluid delivery line in the rigid probe and onto the tissue. The system is able to deliver dye volumes with 50-nL precision.
The mobile cart, shown in Figure 2 , contains the optical scan unit, laser, computer, and primary operator console. Live images are visible from the surgical field on a secondary display. The entire mobile unit can be moved into a surgical suite and set up to image within a few minutes.
To collect images, the microlaparoscope illuminates the tissue with laser light. The laser illumination excites the locally delivered contrast agent. Fluorescent signal emitted in the tissue is then collected by the miniature objective lens. The lens images the fluorescent signal onto the distal face of the coherent fiber-optic bundle. This signal is then relayed back to the optical scan unit. The optical scan unit is a high-speed confocal microscope. It contains the laser, scan mirrors, optics, confocal aperture, and detector. Detailed technical descriptions of the miniature objective lens, the optical scan unit, and the confocal microlaparoscope system are discussed elsewhere.
Fluorescent contrast agents
Imaging with the microlaparoscope requires the application of an exogenous fluorescent contrast agent. Fluorescein sodium is a dye approved for use in humans for retinal angiography and was selected as the contrast agent for initial testing of the confocal microlaparoscope. The clinical study was conducted using approximately 1–3 μL of 1% fluorescein sodium per imaging site. This volume is sufficient for staining a 5-mm diameter area on the tissue surface. The localized application of fluorescein sodium also marks the imaging site for correlated biopsies.
Topically applied fluorescein sodium binds rather nonspecifically to proteins in tissue. This results in low-contrast images that have limited diagnostic value for detecting ovarian cancer. The ovaries were also imaged using acridine orange (AO) following extraction from the patient. AO is a nuclear stain and an excellent fluorophore for visualizing cellular distributions. Its diagnostic potential has been demonstrated previously in the context of detecting ovarian cancer.
Sites on the ovary that were imaged with fluorescein sodium during the in vivo procedure were identified using a UV flashlight. At nearby locations, approximately 1–3 μL of 330-μM/L AO was applied to the surface of the excised ovary and imaged with the microlaparoscope. These sites were biopsied and processed with standard histopathologic procedures using hematoxylin and eosin (H&E) staining.
Toward the end of the pilot study, AO was used in vivo to validate that the same contrast observed ex vivo could be obtained in vivo. For in vivo testing, 1 μL of 330-μM/L AO was used to image the ovaries following a protocol that prevented any other organ from coming into contact with the dye. This was accomplished by imaging the ovary inside an endobag. Food and Drug Administration approval to use AO in this context was granted under investigational new drug 102603.
Confocal imaging procedure
The pilot study involved 21 patients imaged over a 1-year time period. All procedures in the study were approved by the institutional review board at the University of Arizona. Participants in the study were recruited at the University Medical Center, Tucson, AZ. Subjects were eligible to participate in the study if they were at least 18 years of age and not pregnant. All subjects were from the cohort of patients undergoing laparoscopic oophorectomy or open surgery. No financial compensation was offered, and there was no diagnostic benefit to the patient for participation in the study.
Standard surgical procedures for clinical oophorectomy were followed, with the addition of microlaparoscope imaging prior to ovary removal. Figure 3 shows the microlaparoscope in use during a clinical procedure. All patients were placed under general endotracheal anesthesia, and abdominal cavities were insufflated with carbon dioxide to raise the abdominal wall. The ovary was partially resected and placed inside an endobag to prevent the patient from being exposed to the contrast agent ( Figure 4 ). The microlaparoscope was inserted through a 5-mm trocar. Conventional laparoscopic image guidance was used to position the microlaparoscope tip into contact with the ovary. Several locations on the surface of the ovary were interrogated using the confocal microlaparoscope. In all patients, the extra imaging with the microlaparoscope was completed in less than 10 minutes.
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