Category: Fluorescence

Fluorescence Spectroscopy

Fluorescence Spectroscopy

Fluorescence spectroscopy is an optical method used to detect and quantify molecules based on their light emission following excitation. It offers high sensitivity, real-time feedback, and molecular specificity, making it foundational to applications in genomics, diagnostics, biosensing, and imaging. From four-color DNA sequencing to targeted theranostics, fluorescence has been widely adopted for both basic research and clinical workflows. The technique has been adapted for use in multimodal diagnostic platforms and embedded within contrast agents for high-resolution imaging. It also plays a role in environmental monitoring and implantable sensors. Work in this area has supported innovations in spectral discrimination, integrated fluorescence into protein-based imaging agents, and extended its use into in vivo physiological monitoring through carbon nanotube–based nanosensors and a fiber-optic benchtop platform for scalable fluorescence detection.

Nine Diagnostics Joins American Cancer Society’s BrightEdge Entrepreneurs Program

Nine Diagnostics Joins American Cancer Society’s BrightEdge Entrepreneurs Program

Nine Diagnostics, a leader in AI-enabled nanosensor technology, has been selected to participate in the American Cancer Society’s BrightEdge Entrepreneurs Program, a highly selective initiative designed to accelerate the most promising oncology-focused startups. This selection marks another significant milestone for Nine Diagnostics as it continues to drive innovation in cancer treatment selection, dosing, optimization, and monitoring.

Nature Nanotechnology: A wavelength-induced frequency filtering method for fluorescent nanosensors in vivo

Nature Nanotechnology: A wavelength-induced frequency filtering method for fluorescent nanosensors in vivo

Fluorescent nanosensors hold the potential to revolutionize life sciences and medicine. However, their adaptation and translation into the in vivo environment is fundamentally hampered by unfavourable tissue scattering and intrinsic autofluorescence. Here we develop wavelength-induced frequency filtering (WIFF) whereby the fluorescence excitation wavelength is modulated across the absorption peak of a nanosensor, allowing the emission signal to be separated from the autofluorescence background, increasing the desired signal relative to noise, and internally referencing it to protect against artefacts. Using highly scattering phantom tissues, an SKH1-E mouse model and other complex tissue types, we show that WIFF improves the nanosensor signal-to-noise ratio across the visible and near-infrared spectra up to 52-fold. This improvement enables the ability to track fluorescent carbon nanotube sensor responses to riboflavin, ascorbic acid, hydrogen peroxide and a chemotherapeutic drug metabolite for depths up to 5.5 ± 0.1 cm when excited at 730 nm and emitting between 1,100 and 1,300 nm, even allowing the monitoring of riboflavin diffusion in thick tissue. As an application, nanosensors aided by WIFF detect the chemotherapeutic activity of temozolomide transcranially at 2.4 ± 0.1 cm through the porcine brain without the use of fibre optic or cranial window insertion. The ability of nanosensors to monitor previously inaccessible in vivo environments will be important for life-sciences research, therapeutics and medical diagnostics.

Implanted Nanosensors in Marine Organisms for Physiological Biologging: Design, Feasibility, and Species Variability

Implanted Nanosensors in Marine Organisms for Physiological Biologging: Design, Feasibility, and Species Variability

In recent decades, biologists have sought to tag animals with various sensors to study aspects of their behavior otherwise inaccessible from controlled laboratory experiments. Despite this, chemical information, both environmental and physiological, remains challenging to collect despite its tremendous potential to elucidate a wide range of animal behaviors. In this work, we explore the design, feasibility, and data collection constraints of implantable, near-infrared fluorescent nanosensors based on DNA-wrapped single-wall carbon nanotubes (SWNT) embedded within a biocompatible poly(ethylene glycol) diacrylate (PEGDA) hydrogel. These sensors are enabled by Corona Phase Molecular Recognition (CoPhMoRe) to provide selective chemical detection for marine organism biologging. Riboflavin, a key nutrient in oxidative phosphorylation, is utilized as a model analyte in in vitro and ex vivo tissue measurements. Nine species of bony fish, sharks, eels, and turtles were utilized on site at Oceanogràfic in Valencia, Spain to investigate sensor design parameters, including implantation depth, sensor imaging and detection limits, fluence, and stability, as well as acute and long-term biocompatibility. Hydrogels were implanted subcutaneously and imaged using a customized, field-portable Raspberry Pi camera system. Hydrogels could be detected up to depths of 7 mm in the skin and muscle tissue of deceased teleost fish ( Sparus aurata and Stenotomus chrysops) and a deceased catshark ( Galeus melastomus). The effects of tissue heterogeneity on hydrogel delivery and fluorescence visibility were explored, with darker tissues masking hydrogel fluorescence. Hydrogels were implanted into a living eastern river cooter ( Pseudemys concinna), a European eel ( Anguilla anguilla), and a second species of catshark ( Scyliorhinus stellaris). The animals displayed no observable changes in movement and feeding patterns. Imaging by high-resolution ultrasound indicated no changes in tissue structure in the eel and catshark. In the turtle, some tissue reaction was detected upon dissection and histopathology. Analysis of movement patterns in sarasa comet goldfish ( Carassius auratus) indicated that the hydrogel implants did not affect swimming patterns. Taken together, these results indicate that this implantable form factor is a promising technique for biologging using aquatic vertebrates with further development. Future work will tune the sensor detection range to the physiological range of riboflavin, develop strategies to normalize sensor signal to account for the optical heterogeneity of animal tissues, and design a flexible, wearable device incorporating optoelectronic components that will enable sensor measurements in moving animals. This work advances the application of nanosensors to organisms beyond the commonly used rodent and zebrafish models and is an important step toward the physiological biologging of aquatic organisms.

Characterization of magnetic nanoparticle-seeded microspheres for magnetomotive and multimodal imaging

Characterization of magnetic nanoparticle-seeded microspheres for magnetomotive and multimodal imaging

Magnetic iron-oxide nanoparticles have been developed as contrast agents in magnetic resonance imaging (MRI) and as therapeutic agents in magnetic hyperthermia. They have also recently been demonstrated as contrast and elastography agents in magnetomotive optical coherence tomography and elastography (MM-OCT and MM-OCE, respectively). Protein-shell microspheres containing suspensions of these magnetic nanoparticles in lipid cores, and with functionalized outer shells for specific targeting, have also been demonstrated as efficient contrast agents for imaging modalities such as MM-OCT and MRI, and can be easily modified for other modalities such as ultrasound, fluorescence, and luminescence imaging. In addition to multimodal contrast-enhanced imaging, these microspheres could serve as drug carriers for targeted delivery under image guidance. Although the preparation and surface modifications of protein microspheres containing iron oxide nanoparticles has been previously described and feasibility studies conducted, many questions regarding their production and properties remain. Since the use of multifunctional microspheres could have high clinical relevance, here we report a detailed characterization of their properties and behavior in different environments to highlight their versatility. The work presented here is an effort for the development and optimization of nanoparticle-based microspheres as multi-modal contrast agents that can bridge imaging modalities on different size scales.

Freddy Nguyen Chosen for an Arnold O. Beckman Postdoctoral Fellows Award

Freddy Nguyen Chosen for an Arnold O. Beckman Postdoctoral Fellows Award

2015 PhD graduate awarded Beckman Postdoc Fellowship – March 30, 2017

Congratulations to Freddy Nguyen, a 2015 Illinois Chemistry PhD graduate, who was chosen for a prestigious Arnold O. Beckman Postdoctoral Fellows Award. Nguyen is a postdoctoral researcher at MIT working on development of nanosensors for in vivomonitoring of cancer therapeutics.

According to Nguyen, “The research I am planning to pursue is focused on the development of nanoscale molecular sensors for probing the tumor and its microenvironment. More specifically, we would like to implant our nanosensors inside tumors to to measure their response at the molecular level to various cancer therapies such as chemotherapeutics and radiation therapy. Our nanosensors are detected using near-infrared fluorescence and Raman spectroscopic techniques allowing us to probe the sensors from a distance using near-infrared light and are not susceptible to photobleaching effects unlike typical endogenous and exogenous fluorophores. These unique features of our nanosensors can allow us with a method to dynamically probe the tumor microenvironment in real-time and in-vivo. Patients currently have to wait until there are measurable size changes on CT or MRI scans or must undergo biopsies of the tumor to determine molecular changes in response to treatment. Having access to that molecularinformation within the first few days of treatment will be a tremendous step forward indetermining whether cancer treatments are working for each patient at a much earlier timeframe than the current standard of care. This allows for the patient and physician to morepromptly manage the treatment of their cancer.”

Optical coherence tomography and targeted multi-modal protein microspheres for cancer imaging

Optical coherence tomography and targeted multi-modal protein microspheres for cancer imaging

The field of biomedical optics has grown quickly over the last two decades as various technological advances have helped increase the acquisition speeds and the sensitivity limits of the technology. During this time, optical coherence tomography (OCT) has been explored for a wide number of clinical applications ranging from cardiology to oncology to primary care. In this thesis, I describe the design and construction of an intraoperative clinical OCT system that can be used to image and classify breast cancer tumor margins as normal, close, or positive. I also demonstrate that normal lymph nodes can be distinguished from reactive or metastatic lymph nodes by looking at the difference in scattering intensity between the cortex and the capsule of the node. Despite the advances of OCT in the detection and diagnosis of breast cancer, this technology is still limited by its field of view and can only provide structural information about the tissue. Structural OCT would benefit from added contrast via sub-cellular or biochemical components via the use of contrast agents and functional OCT modalities. As with most other optical imaging techniques, there is a trade off between the imaging field of view and the high-resolution microscopic imaging. In this thesis, I demonstrate for the first time that MM-OCT can be used as a complimentary technique to wide field imaging modalities, such as magnetic resonance imaging (MRI) or fluorescence imaging, using targeted multi-modal protein microspheres. By using a single contrast agent to bridge the wide field and microscopic imaging modalities, a wide field imaging technique can be used to initially localize the contrast agent at the site of interest to guide the location of the MM-OCT imaging to provide a microscopic view. In addition to multi-modal contrast, the microspheres were functionalized with RGD peptides that can target various cancer cell lines. The cancer cells readily endocytosed bound protein microspheres, revealing the possibility that these protein microspheres could also be used as therapeutic agents. These investigations furthered the utility of the OCT technology for cancer imaging and diagnosis.

Targeted multi-modal protein microspheres for cancer imaging

Targeted multi-modal protein microspheres for cancer imaging

Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications from ophthalmology to oncology. OCT is analogous to ultrasound but with micron-scale resolution by using light waves instead of sound waves to provide detailed structural information at the cellular level. The development of contrast agents has been critical to the development of OCT and its functional modalities such as magneto-motive OCT (MM-OCT). MM-OCT is a modality of OCT in which a small external magnetic field is modulated on and off during imaging, providing an added dimension of contrast from the magnetic particle responses. Protein microspheres consisting of a hydrophobic oil core and a hydrophilic BSA protein shell provide the vehicle for a multi-modal contrast agent. The microspheres encapsulate iron oxide nanoparticles in the oil core, providing magnetic signal contrast, and dyes such as Nile Red and DiR iodide, providing fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Using dynamic light scattering, we found the size distribution of the microspheres to be between 1-5 µm. Under SEM and TEM, we were able to visualize the various layers and coatings, such as silica and RGD peptides, of the microsphere. The microspheres were optimized to maximize the magnetic contrast under MM-OCT and MRI, and the fluorescent contrast under a dark box fluorescence imaging system, and fluorescence microscopy. These studies validated the use of MM-OCT as a method for quantifying the relative amount of iron oxide and the relative number of microspheres in the samples. To address the binding specificity and sensitivity of the RGD coated microspheres to the integrin receptors, the microspheres were incubated with cell lines of varying expression levels of the alpha(v)beta(3) integrin receptor and visualized under fluorescence microscopy. The cell lines used in this study included a normal epithelial cell line: hTERT-HME1, and several human breast cancer cell lines: HCC38, SK-BR-3, MCF-7, ZR-75-1, MDA-MB-231, and MDA-MB-435S. These results were externally validated by quantification of the receptors using indirect immunohistochemical staining and flow cytometry. Preliminary results, using the multi-spectral dark box fluorescence imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor and to lymph nodes. This is highly suggestive of the microsphere’s selective binding to the vasculature. By combining the benefits of these various imaging modalities in a single agent, it becomes possible to use a wide-field imaging method such as MRI or small animal fluorescence imaging to initially locate the agents in-vivo, to use MM-OCT to provide micron scale resolution structural images in-vivo, and to use fluorescence microcopy to confirm the localization of these particles ex-vivo.