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.

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 Endoscopy with Light Gating for Early Cancer Detection.

This paper reports the application of endoscopic light scattering spectroscopy (LSS) with light gating to detect malignancies in the biliary and pancreatic ducts, and also reviews the application of endoscopic LSS for differentiating cystic neoplasms in the pancreas and detecting invisible dysplasia in Barrett's esophagus. Information about tissue structure within the superficial epithelium where malignancy starts is present within the spectra of reflected light. Fortunately, this component of the reflected light is not yet randomized. However multiple scattering randomizes the signal from the underlying connective tissue which obscures the desired signal. In order to extract diagnostic information from the reflected signal the multiple scattering component related to connective tissue scattering and absorption must be removed. This is accomplished using described here spatial or polarization gating implemented with endoscopically compatible fiber optic probes.

Picoanalysis of Drugs in Biofluids with Quantitative Label-Free Surface-Enhanced Raman Spectroscopy.

The enormous increase of Raman signal in the vicinity of metal nanoparticles allows surface-enhanced Raman spectroscopy (SERS) to be employed for label-free detection of substances at extremely low concentrations. However, the ultimate potential of label-free SERS to identify pharmaceutical compounds at low concentrations, especially in relation to biofluid sensing, is far from being fully realized. Opioids are a particular challenge for rapid clinical identification because their molecular structural similarities prevent their differentiation with immunolabeling approaches. In this paper, a new method called quantitative label-free SERS (QLF-SERS) which involves the formation of halide-conjugated gold nanoclusters trapping the analyte of interest near the SERS hot spots is reported, and it is demonstrated that it yields a 105 fold improvement in the detection limit over previously reported results for the entire class of clinically relevant opioids and their metabolites. Measurements of opioid concentrations in multicomponent mixtures are also demonstrated. QLF-SERS has comparable detection limits as currently existing laboratory urine drug testing techniques but is significantly faster and inexpensive and, therefore, can be easily adapted as part of a rapid clinical laboratory routine.

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.

Light scattering spectroscopy identifies the malignant potential of pancreatic cysts during endoscopy.

Pancreatic cancers are usually detected at an advanced stage and have poor prognosis. About one fifth of these arise from pancreatic cystic lesions. Yet not all lesions are precancerous, and imaging tools lack adequate accuracy for distinguishing precancerous from benign cysts. Therefore, decisions on surgical resection usually rely on endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Unfortunately, cyst fluid often contains few cells, and fluid chemical analysis lacks accuracy, resulting in dire consequences, including unnecessary pancreatic surgery for benign cysts and the development of cancer. Here, we report an optical spectroscopic technique, based on a spatial gating fibre-optic probe, that predicts the malignant potential of pancreatic cystic lesions during routine diagnostic EUS-FNA procedures. In a double-blind prospective study in 25 patients, with 14 cysts measured in vivo and 13 postoperatively, the technique achieved an overall accuracy of 95%, with a 95%confidence interval of 78-99%, in cysts with definitive diagnosis.

Spectroscopy of scattered light for the characterization of micro and nanoscale objects in biology and medicine.

The biomedical uses for the spectroscopy of scattered light by micro and nanoscale objects can broadly be classified into two areas. The first, often called light scattering spectroscopy (LSS), deals with light scattered by dielectric particles, such as cellular and sub-cellular organelles, and is employed to measure their size or other physical characteristics. Examples include the use of LSS to measure the size distributions of nuclei or mitochondria. The native contrast that is achieved with LSS can serve as a non-invasive diagnostic and scientific tool. The other area for the use of the spectroscopy of scattered light in biology and medicine involves using conducting metal nanoparticles to obtain either contrast or electric field enhancement through the effect of the surface plasmon resonance (SPR). Gold and silver metal nanoparticles are non-toxic, they do not photobleach, are relatively inexpensive, are wavelength-tunable, and can be labeled with antibodies. This makes them very promising candidates for spectrally encoded molecular imaging. Metal nanoparticles can also serve as electric field enhancers of Raman signals. Surface enhanced Raman spectroscopy (SERS) is a powerful method for detecting and identifying molecules down to single molecule concentrations. In this review, we will concentrate on the common physical principles, which allow one to understand these apparently different areas using similar physical and mathematical approaches. We will also describe the major advancements in each of these areas, as well as some of the exciting recent developments.

Rapid optimization of metal nanoparticle surface modification with high-throughput gel electrophoresis.

The ability to effectively control and optimize surface modification of metal nanoparticles is paramount to the ability to employ metal nanoparticles as diagnostic and therapeutic agents in biology and medicine. Here we present a high-throughput two-dimensional-grid gel electrophoresis cell (2D-GEC)-based method, capable of optimizing the surface modification of as many as 96 samples of metal nanoparticles in approximately 1 h. The 2D-GEC method determines not only the average zeta-potential of the modified particles but also the homogeneity of the surface modification by measuring the distance between the front of the sample track and the area where the maximum optical density is achieved. The method was tested for optimizing pH and concentration of the modifiers (pM) for functionalizing gold nanorod thiol-containing acidic agents.

Spectral Imaging with Scattered Light: From Early Cancer Detection to Cell Biology.

This article reports the evolution of scanning spectral imaging techniques using scattered light for minimally invasive detection of early cancerous changes in tissue and cell biology applications. Optical spectroscopic techniques have shown promising results in the diagnosis of disease on a cellular scale. They do not require tissue removal, can be performed in vivo, and allow for real time diagnoses. Fluorescence and Raman spectroscopy are most effective in revealing molecular properties of tissue. Light scattering spectroscopy (LSS) relates the spectroscopic properties of light elastically scattered by small particles, such as epithelial cell nuclei and organelles, to their size, shape and refractive index. It is capable of characterizing the structural properties of tissue on cellular and sub-cellular scales. However, in order to be useful in the detection of early cancerous changes which are otherwise not visible to the naked eye, it must rapidly survey a comparatively large area while simultaneously detecting these cellular changes. Both goals are achieved by combining LSS with spatial scanning imaging. Two examples are described in this article. The first reviews a clinical system for screening patients with Barrett's esophagus. The second presents a novel advancement in confocal light absorption and scattering spectroscopic (CLASS) microscopy.

Photon diffusion near the point-of-entry in anisotropically scattering turbid media.

From astronomy to cell biology, the manner in which light propagates in turbid media has been of central importance for many decades. However, light propagation near the point-of-entry in turbid media has never been analytically described, until now. Here we report a straightforward and accurate method that overcomes this longstanding, unsolved problem in radiative transport. Our theory properly treats anisotropic photon scattering events and takes the specific form of the phase function into account. As a result, our method correctly predicts the spatially dependent diffuse reflectance of light near the point-of-entry for any arbitrary phase function. We demonstrate that the theory is in excellent agreement with both experimental results and Monte Carlo simulations for several commonly used phase functions.

Multispectral scanning during endoscopy guides biopsy of dysplasia in Barrett's esophagus.

Esophageal cancer is increasing in frequency in the United States faster than any other cancer. Barrett's esophagus, an otherwise benign complication of esophageal reflux, affects approximately three million Americans and precedes almost all cases of esophageal cancer. If detected as high-grade dysplasia (HGD), most esophageal cancers can be prevented. Standard-of-care screening for dysplasia uses visual endoscopy and a prescribed pattern of biopsy. This procedure, in which a tiny fraction of the affected tissue is selected for pathological examination, has a low probability of detection because dysplasia is highly focal and visually indistinguishable. We developed a system called endoscopic polarized scanning spectroscopy (EPSS), which performs rapid optical scanning and multispectral imaging of the entire esophageal surface and provides diagnoses in near real time. By detecting and mapping suspicious sites, guided biopsy of invisible, precancerous dysplasia becomes practicable. Here we report the development of EPSS and its application in several clinical cases, one of which merits special consideration.

Gold nanorod light scattering labels for biomedical imaging.

Gold nanorods can be used as extremely bright labels for differential light scattering measurements using two closely spaced wavelengths, thereby detecting human disease through several centimeters of tissue in vivo. They have excellent biocompatibility, are non-toxic, and are not susceptible to photobleaching. They have narrow, easily tunable plasmon spectral lines and thus can image multiple molecular targets simultaneously. Because of their small size, gold nanorods can be transported to various tissues inside the human body via the vasculature and microvasculature, and since they are smaller than vascular pore sizes, they can easily cross vascular space and enter individual cells.

Diagnostic imaging of esophageal epithelium with clinical endoscopic polarized scanning spectroscopy instrument.

This letter reports the development of an endoscopic polarized scanning spectroscopy (EPSS) instrument compatible with existing endoscopes. This instrument uses light scattering spectroscopy (LSS). In proof-of-principle studies using a single-point instrument, LSS has successfully demonstrated the ability to identify pre-cancer in the epithelial tissues of five different organs, including Barrett's esophagus (BE). The EPSS instrument can provide real time in vivo information on the location of otherwise invisible high grade dysplasia (HGD), a predictor of adenocarcinoma, and thus can serve as a guide for biopsy. It should greatly reduce the time and labor involved in performing screening and obtaining diagnoses, cause less patient discomfort and ensure that fewer biopsies are required for the reliable location of pre-cancerous lesions.

Light-scattering spectroscopy differentiates fetal from adult nucleated red blood cells: may lead to noninvasive prenatal diagnosis.

Present techniques for prenatal diagnosis are invasive and present significant risks of fetal loss. Noninvasive prenatal diagnosis utilizing fetal nucleated red blood cells (fNRBC) circulating in maternal peripheral blood has received attention, since it poses no risk to the fetus. However, because of the failure to find broadly applicable identifiers that can differentiate fetal from adult NRBC, reliable detection of viable fNRBC in amounts sufficient for clinical use remains a challenge. In this Letter we show that fNRBC light-scattering spectroscopic signatures may lead to a clinically useful method of minimally invasive prenatal genetic testing.

Confocal light absorption and scattering spectroscopic microscopy monitors organelles in live cells with no exogenous labels.

This article reports the development of an optical imaging technique, confocal light absorption and scattering spectroscopic (CLASS) microscopy, capable of noninvasively determining the dimensions and other physical properties of single subcellular organelles. CLASS microscopy combines the principles of light-scattering spectroscopy (LSS) with confocal microscopy. LSS is an optical technique that relates the spectroscopic properties of light elastically scattered by small particles to their size, refractive index, and shape. The multispectral nature of LSS enables it to measure internal cell structures much smaller than the diffraction limit without damaging the cell or requiring exogenous markers, which could affect cell function. Scanning the confocal volume across the sample creates an image. CLASS microscopy approaches the accuracy of electron microscopy but is nondestructive and does not require the contrast agents common to optical microscopy. It provides unique capabilities to study functions of viable cells, which are beyond the capabilities of other techniques.

Confocal light absorption and scattering spectroscopic microscopy.

We have developed a novel optical method for observing submicrometer intracellular structures in living cells, which is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light-scattering spectroscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales of the order of 100 nm.