In vivo detection of bile duct pre-cancer with endoscopic light scattering spectroscopy.

Bile duct cancer is the second most common primary liver cancer, with most diagnoses occurring in the advanced stages. This leads to a poor survival rate, which means a technique capable of reliably detecting pre-cancer in the bile duct is urgently required. Unfortunately, radiological imaging lacks adequate accuracy for distinguishing dysplastic and benign biliary ducts, while endoscopic techniques, which can directly assess the bile duct lining, often suffer from insufficient sampling. Here, we report an endoscopic optical light scattering technique for clinical evaluation of the malignant potential of the bile duct. This technique employs an ultraminiature spatial gating fiber optic probe compatible with cholangioscopes and endoscopic retrograde cholangiopancreatography (ERCP) catheters. The probe allowed us to investigate the internal cellular composition of the bile duct epithelium with light scattering spectroscopy (LSS) and phenotypic properties of the underlying connective tissue with diffuse reflectance spectroscopy (DRS). In a pilot in vivo double-blind prospective study involving 29 patients undergoing routine ERCP procedures, the technique detected malignant transformation with 97% accuracy, showing that biliary duct pre-cancer can be reliably identified in vivo non-invasively.

Biopsy channel of the endoscope as a potential source of infectious droplets during GI endoscopy.

BACKGROUND AND AIMS: During endoscopy, droplets with the potential to transmit infectious diseases are known to emanate from a patient's mouth and anus, but they may also be expelled from the biopsy channel of the endoscope. The main goal of our study was to quantify droplets emerging from the biopsy channel during clinical endoscopy. METHODS: A novel light-scattering device was used to measure droplets emanating from the biopsy channel. An endoscopy model was created, and in vitro measurements were carried out during air insufflation, air and water suctioning, and the performance of biopsy sampling. Similar measurements were then made on patients undergoing endoscopy, with all measurements taking place over 2 days to minimize variation. RESULTS: During in vitro testing, no droplets were observed at the biopsy channel during air insufflation or air and water suctioning. In 3 of 5 cases, droplets were observed during biopsy sampling, mostly when the forceps were being removed from the endoscope. In the 22 patients undergoing routine endoscopy, no droplets were observed during air insufflation and water suctioning. Droplets were detected in 1 of 11 patients during air suctioning. In 9 of 18 patients undergoing biopsy sampling and 5 of 6 patients undergoing snare polypectomies, droplets were observed at the biopsy channel, mostly when instruments were being removed from the endoscope. CONCLUSIONS: We found that the biopsy channel may be a source of infectious droplets, especially during the removal of instruments from the biopsy channel. When compared with droplets reported from the mouth and anus, these droplets were larger in size and therefore potentially more infectious.

Rapid detection and identification of bacteria directly from whole blood with light scattering spectroscopy based biosensor.

Bacterial infections are one of the major causes of death worldwide. The identification of a bacterial species that is the source of an infection generally takes a long time, and often exceeds the treatment window for seriously ill patients. Many of these deaths are preventable if the bacterial species can be identified quickly. Here we present an optical spectroscopic method for rapid detection and identification of bacteria directly from whole blood using a light scattering spectroscopy technique. This technique was originally developed to detect pre-cancerous changes in epithelial tissues, characterize changes in tissue on the cellular scale, and characterize biological structures comparable to or smaller than a single wavelength. We demonstrate here that not only can an inexpensive light scattering spectroscopy-based biosensor rapidly detect and identify four bacteria species in the blood, responsible for the majority of death causing infections, but that species-level identification can potentially be made based on approximately one thousand bacterial cells per milliliter of blood. Observing entire colonies or performing susceptibility testing is therefore not required.

Coherent confocal light scattering spectroscopic microscopy evaluates cancer progression and aggressiveness in live cells and tissue.

The observation of biological structures in live cells beyond the diffraction limit with super-resolution fluorescence microscopy is limited by the ability of fluorescence probes to permeate live cells and the effect of these probes, which are often toxic, on cellular behavior. Here we present a coherent confocal light scattering and absorption spectroscopic microscopy that for the first time enables the use of large numerical aperture optics to characterize structures in live cells down to 10 nm spatial scales, well beyond the diffraction limit. Not only does this new capability allow high resolution microscopy with light scattering contrast, but it can also be used with almost any light scattering spectroscopic application which employs lenses. We demonstrate that the coherent light scattering contrast based technique allows continuous temporal tracking of the transition from non-cancerous to an early cancerous state in live cells, without exogenous markers. We also use the technique to sense differences in the aggressiveness of cancer in live cells and for label free identification of different grades of cancer in resected tumor tissues.

Spectroscopic label-free microscopy of changes in live cell chromatin and biochemical composition in transplantable organoids.

Organoids formed from human induced pluripotent stem cells (hiPSCs) could be a limitless source of functional tissue for transplantations in many organs. Unfortunately, fine-tuning differentiation protocols to form large quantities of hiPSC organoids in a controlled, scalable, and reproducible manner is quite difficult and often takes a very long time. Recently, we introduced a new approach of rapid organoid formation from dissociated hiPSCs and endothelial cells using microfabricated cell-repellent microwell arrays. This approach, when combined with real-time label-free Raman spectroscopy of biochemical composition changes and confocal light scattering spectroscopic microscopy of chromatin transition, allows for monitoring live differentiating organoids without the need to sacrifice a sample, substantially shortening the time of protocol fine-tuning. We used this approach to both culture and monitor homogeneous liver organoids that have the main functional features of the human liver and which could be used for cell transplantation liver therapy in humans.

Two-photon polymerization nanofabrication of ultracompact light scattering spectroscopic probe for detection of pre-cancer in pancreatic cyst.

Pancreatic cancer has one of the worst survival rates of all major cancers, with pancreatic cystic lesions accounting for one in three pancreatic surgeries. The current gold-standard for diagnosis of pancreatic cyst malignancy is based on the endoscopic ultrasound guided fine-needle aspiration (EUS-FNA) procedure, which suffers from a low accuracy in detecting malignancy. Here we present the design and two-photon polymerization based fabrication of refractive and reflective non-contact probes, capable of rapid surveillance of the entire internal cyst surface-an advance over the contact probe we recently developed that allowed, for the first time, reliable evaluation of pancreatic cyst malignant potential in vivo. We employed a novel two-photon polymerization technique, which allows direct laser-writing to an accuracy of tens of nanometers, to fit the probe within the 540 micrometer internal diameter EUS-FNA needle. The newly constructed probes show excellent separation of the illumination and collection beams, essential for proper operation of the spatial gating method. These probes can be used clinically to perform rapid "optical biopsy", ultimately eliminating unnecessary pancreatic surgeries on benign cysts and dangerous delays in surgical removal of malignant cysts, improving patient prognosis and quality of life.

Multispectral light scattering endoscopic imaging of esophageal precancer.

Esophageal adenocarcinoma is the most rapidly growing cancer in America. Although the prognosis after diagnosis is unfavorable, the chance of a successful outcome increases tremendously if detected early while the lesion is still dysplastic. Unfortunately, the present standard-of-care, endoscopic surveillance, has major limitations, since dysplasia is invisible, often focal, and systematic biopsies typically sample less than one percent of the esophageal lining and therefore easily miss malignancies. To solve this problem we developed a multispectral light scattering endoscopic imaging system. It surveys the entire esophageal lining and accurately detects subcellular dysplastic changes. The system combines light scattering spectroscopy, which detects and identifies invisible dysplastic sites by analyzing light scattered from epithelial cells, with rapid scanning of the entire esophageal lining using a collimated broadband light beam delivered by an endoscopically compatible fiber optic probe. Here we report the results of the first comprehensive multispectral imaging study, conducted as part of routine endoscopic procedures performed on patients with suspected dysplasia. In a double-blind study that characterized the system's ability to serve as a screening tool, 55 out of 57 patients were diagnosed correctly. In addition, a smaller double-blind comparison of the multispectral data in 24 patients with subsequent pathology at locations where 411 biopsies were collected yielded an accuracy of 90% in detecting individual locations of dysplasia, demonstrating the capability of this method to serve as a guide for biopsy.