Clinical detection of premalignant lesions often requires labeling with molecular probes. In the gastrointestinal tract, most cancers originate in epithelial layers which are interrogated using topically-applied probes with conjugated fluorescence dyes. As part of clinical trials using fluorescence peptide probes, we have developed two different instruments that rapidly image the biopsy at the bedside before fixation. These multimodal images (visible light reflectance and near infrared fluorescence) provide verification that the targeted lesion was sampled, and provide feedback to the clinician at the bedside for any follow-up procedure. Performance of these two prototypes are compared for fluorescence sensitivity and multimodal image quality.
Although a greater extent of tumor resection is important for patients’ survival, complete tumor removal, especially tumor margins, remains challenging due to the lack of sensitivity and specificity of current surgical guidance techniques at the margins. Intraoperative fluorescence imaging with targeted fluorophores is promising for tumor margin delineation. To verify the tumor margins detected by the fluorescence images, it is necessary to register fluorescence with histological images, which provide the ground truth for tumor regions. However, current registration methods compare fluorescence images to a single-layer histological slide, which is selected subjectively and represents a single plane of the three-dimensional tumor. A multistep pipeline is established to correlate fluorescence images to stacked histological images, including fluorescence calibration and multistep registration. Multiple histological slices are integrated as a two-dimensional (2-D) tumor map using optical attenuation model and average intensity projection. A BLZ-100-labeled medulloblastoma mouse model is used to test the whole framework. On average, the synthesized 2-D tumor map outperforms the selected best slide as ground truth [Dice similarity coefficient (DSC): 0.582 versus 0.398, with significant differences; mean area under the curve (AUC) of the receiver operating characteristic curve: 88% versus 85.5%] and the randomly selected slide as ground truth (DSC: 0.582 versus 0.396 with significant differences; mean AUC: 88% versus 84.1% with significant differences), which indicates our pipeline is reliable and can be applied to investigate targeted fluorescence probes in tumor margin detection. Following this proposed pipeline, BLZ-100 shows enhancement in both tumor cores and tumor margins (mean target-to-background ratio: 8.64 ± 5.76 and 4.82 ± 2.79, respectively).
Fluorescence endoscopy is emerging for guiding biopsy and early cancer detection in the gastrointestinal tract. A multimodal scanning fiber endoscope (mmSFE) with 2 fluorescence labeled peptides is designed to image overexpressed biomarkers associated with esophageal adenocarcinoma (EAC) and high-grade dysplasia (HGD), thus detecting early neoplasia. Quantification of multiplexed fluorescence images is critical, which ‘red-flags’ suspicious regions and supports diagnosis. The Target/Background (T/B) ratio is calculated to quantitatively evaluate fluorescence images. However T/B ratios are based on fluorescence intensities alone, so adding morphological features can be critical to providing evidence for diagnosis. Moreover, currently the T/B ratio is calculated for a single fluorescence channel. A protocol for multiplexed fluorescence quantification is needed.
Materials and Methods: Peptides targeting EGFR and ErbB2 are labeled with NIR fluorescence Cy5 and IRdye800, respectively. A mmSFE with 2.4mm flexible shaft and wide-field forward-view imaging is designed to image these two near-infrared fluorophores with an additional reflectance channel for anatomical identification. In first-in-human clinical trials the two peptides are topically sprayed, briefly incubated, and then rinsed in subjects at high risk of EAC. Both fluorescence channels were captured simultaneously with the co-registered reflectance channel at 30Hz. After removing artifacts, frames were manually selected using morphological features. T/B ratios were then calculated for all fluorescence channels in selected frames.
Results and Conclusions: T/B ratios for HGD subjects are greater than for healthy subjects in at least one of the fluorescence channels. Future work will design algorithms to automatically select suspicious regions based on morphological features. More analysis will be performed based on co-registered fluorescence channels.
Multimodal endoscopy, with fluorescence labeled peptides specific for multiple biomarkers, is a promising technique to detect early-stage GI tract cancers in vivo. A reproducible bile duct phantom with near infrared (NIR) fluorescent targets is developed for practicing the clinical study protocol and quantitative evaluation of multimodal endoscope performance during biliary duct imaging. Furthermore, this phantom with strictures will be used for testing new fluorescence guided biopsy devices. Materials and Methods: A soft, flexible synthetic human bile duct was fabricated from a paintable silicone rubber. Due to the complex structure of biliary system (such as hepatic ducts, cystic duct, and common bile duct), the template mold for lumen was designed for 3D printing using Polyvinyl Acetate (PVA), which can be later dissolved in water. Cured gelatin patches with different concentrations of fluorescence dyes (Cy5 and IRDye800) were placed onto the mold. Then silicone rubber with pigments to simulate visual appearance was painted in layers. After the silicone curing, the phantom was placed in warm water (40 degreeC) to dissolve PVA. Two different multimodal scanning fiber endoscope systems, RGB reflectance + NIR fluorescence and 3 fluorescence (IRDye800, Cy5, and FITC) + grayscale reflectance, were used to test the phantom. Results: The bile duct phantom is flexible, stable, and repeatable to fabricate with strictures. Clinical study procedures of fluorescence labeling were evaluated quantitatively. The NIR fluorescence targets in the phantom were used to calibrate the imaging system, further develop image-based biomarker quantification.
Minimally-invasive optical imaging is being advanced by molecular probes that enhance contrast using fluorescence. The applications in cancer imaging are very broad, ranging from early diagnosis of cancer to the guiding of interventions, such as surgery. The high-sensitivity afforded by wide-field fluorescence imaging using scanning laser light is being developed for these broad applications. The platform technology being introduced for fluorescence-guided surgery is multimodal scanning fiber endoscope (mmSFE), which places a sub-1-mm optical fiber scanner at the tip of a highly flexible scope. Because several different laser wavelengths can be mixed and scanned together, full-color reflectance imaging can be combined with near infrared (NIR) fluorescence imaging in a new 4-channel multimodal SFE. Different imaging display modes are evaluated to provide surgeons fluorescence information with anatomical background preserved. These preliminary results provide a measure of mmSFE imaging performance in vitro and ex vivo, using a mouse model of brain cancer and BLZ-100 fluorescence tumor indicator. The mmSFE system generated wide-field 30 Hz video of concurrent reflectance and NIR fluorescence with sensitivity below 1 nM in vitro. Using the ex vivo mouse brain tumor model, the low-power 785-nm laser source does not produce any noticeable photobleaching of tumors with strong fluorescence signal over 30 minutes of continuous multimodal imaging. The wide-field NIR fluorescence images of the mouse brain surface produced a match to the conventional histology slices by processing the hematoxylin signal in a mean intensity projection to the outer surface and then registering with the mmSFE image. These results indicate the potential for the mmSFE and BLZ-100 tumor indicator for fluorescence guidance of keyhole neurosurgery.
Multimodal endoscopy using fluorescence molecular probes is a promising method of surveying the entire esophagus to detect cancer progression. Using the fluorescence ratio of a target compared to a surrounding background, a quantitative value is diagnostic for progression from Barrett’s esophagus to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). However, current quantification of fluorescent images is done only after the endoscopic procedure. We developed a Chan–Vese-based algorithm to segment fluorescence targets, and subsequent morphological operations to generate background, thus calculating target/background (T/B) ratios, potentially to provide real-time guidance for biopsy and endoscopic therapy. With an initial processing speed of 2 fps and by calculating the T/B ratio for each frame, our method provides quasireal-time quantification of the molecular probe labeling to the endoscopist. Furthermore, an automatic computer-aided diagnosis algorithm can be applied to the recorded endoscopic video, and the overall T/B ratio is calculated for each patient. The receiver operating characteristic curve was employed to determine the threshold for classification of HGD/EAC using leave-one-out cross-validation. With 92% sensitivity and 75% specificity to classify HGD/EAC, our automatic algorithm shows promising results for a surveillance procedure to help manage esophageal cancer and other cancers inspected by endoscopy.
For patients with malignant brain tumors (glioblastomas), a safe maximal resection of tumor is critical for an
increased survival rate. However, complete resection of the cancer is hard to achieve due to the invasive nature
of these tumors, where the margins of the tumors become blurred from frank tumor to more normal brain tissue,
but in which single cells or clusters of malignant cells may have invaded. Recent developments in fluorescence
imaging techniques have shown great potential for improved surgical outcomes by providing surgeons
intraoperative contrast-enhanced visual information of tumor in neurosurgery. The current near-infrared (NIR)
fluorophores, such as indocyanine green (ICG), cyanine5.5 (Cy5.5), 5-aminolevulinic acid (5-ALA)-induced
protoporphyrin IX (PpIX), are showing clinical potential to be useful in targeting and guiding resections of such
tumors. Real-time tumor margin identification in NIR imaging could be helpful to both surgeons and patients by
reducing the operation time and space required by other imaging modalities such as intraoperative MRI, and has
the potential to integrate with robotically assisted surgery. In this paper, a segmentation method based on the
Chan-Vese model was developed for identifying the tumor boundaries in an ex-vivo mouse brain from relatively
noisy fluorescence images acquired by a multimodal scanning fiber endoscope (mmSFE). Tumor contours were
achieved iteratively by minimizing an energy function formed by a level set function and the segmentation
model. Quantitative segmentation metrics based on tumor-to-background (T/B) ratio were evaluated. Results
demonstrated feasibility in detecting the brain tumor margins at quasi-real-time and has the potential to yield
improved precision brain tumor resection techniques or even robotic interventions in the future.
Multimodal endoscopy, with fluorescence-labeled probes binding to overexpressed molecular targets, is a promising technology to visualize early-stage cancer. T/B ratio is the quantitative analysis used to correlate fluorescence regions to cancer. Currently, T/B ratio calculation is post-processing and does not provide real-time feedback to the endoscopist. To achieve real-time computer assisted diagnosis (CAD), we establish image processing protocols for calculating T/B ratio and locating high-risk fluorescence regions for guiding biopsy and therapy in Barrett’s esophagus (BE) patients.
Methods: Chan-Vese algorithm, an active contour model, is used to segment high-risk regions in fluorescence videos. A semi-implicit gradient descent method was applied to minimize the energy function of this algorithm and evolve the segmentation. The surrounding background was then identified using morphology operation. The average T/B ratio was computed and regions of interest were highlighted based on user-selected thresholding. Evaluation was conducted on 50 fluorescence videos acquired from clinical video recordings using a custom multimodal endoscope.
Results: With a processing speed of 2 fps on a laptop computer, we obtained accurate segmentation of high-risk regions examined by experts. For each case, the clinical user could optimize target boundary by changing the penalty on area inside the contour.
Conclusion: Automatic and real-time procedure of calculating T/B ratio and identifying high-risk regions of early esophageal cancer was developed. Future work will increase processing speed to <5 fps, refine the clinical interface, and apply to additional GI cancers and fluorescence peptides.
Optical imaging modalities and therapy monitoring protocols are required for the emergence of non-surgical interventions for treating infections in teeth to remineralize the enamel. Current standard of visual inspection, tactile probing and radiograph for caries detection is not highly sensitive, quantitative, and safe. Furthermore, the latter two are not viable options for interproximal caries. We present preliminary results of multimodal laser-based imaging and uorescence spectroscopy in a blinded clinical study comparing two topical therapies of early interproximal caries in children. With a spacer placed interproximally both at baseline and followup examinations, the 405-nm excited red porphyrin uorescence imaging with green auto uorescence is measured and compared to a 12-month follow-up. 405-nm laser-induced uorescence spectroscopy is also measured from the center of selected multimodal video imaging frames. These results of three subjects are analyzed both qualitatively by comparing spectra and quantitatively based on uorescence region segmentation, and then are compared to the standard of care(visual examination and radiograph interpretation). Furthermore, this study points out challenges associated with optically monitoring non-surgical dental interventions over long periods of time in clinical practice and also indicates future direction for improvement on the protocol.
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