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| Members (L-R) Josh Pfefer, Anant Agrawal, Ilko Ilev, David Royston, Stephanie Matchette, Ron Waynant |
Minimally invasive photonic biosensor techniques are potential alternatives to conventional medical methods for diagnosis of diseases. These techniques offer an effective, fast and painless way for sensing and monitoring of various biomedical quantities. Over the past several years, progress in nanotechnology has led to the development of novel optical nanobiosensors, which are sensors with dimensions on the nanometer scale. This has opened up new horizons for single cell and intracellular sensing and measurements. Cellular and intracellular light-tissue interaction mechanisms and photochemical processes need to be understood in order to optimize effectiveness and ensure the safety of laser therapy, photodynamic treatment, cell-microbe interactions, and microbial ecology. The project objective is focused on the study of fundamental light-tissue interaction mechanisms at the cellular and intracellular level using fiber-optic nanobiosensor probes equipped with tapered nanometer scale sensing tips. The nanobiosensors will be utilized for direct probing and chemical analysis within individual cells and within the subcellular organelles. In this way we will be able to detect small concentrations of target molecules or intracellular analytes such as reactive oxygen species, calcium, and glucose. To realize the project ideas we will apply various experimental approaches and methods (see Figs. 1 and 2) including direct optical spectroscopy, time-correlated single photon counting method, smart fiber-optic sensor probes, and high-resolution imaging techniques. The results of the study are important to understanding basic medical processes including: the process of photodynamic cancer cell killing, the introduction of cancer causing environmental agents through the epidermal barrier, the generation of beneficial chemicals and cellular repair, and the effects of light activated oxygen. The mechanisms of light-tissue interactions identified by the experiments could play a pivotal role in determining safety and effectiveness both in laser and photodynamic therapy.
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| Figure 1. Principal design of a combined (a dual-nanosensor-probe setup and a near-field configuration) technique for direct optical spectroscopy of single cells and intracellular components |
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| Figure 2. All-optical-waveguide laser delivery system using a direct grazing-incidence-based hollow taper and a smart tissue-activated fiber probe |
Despite the broad range of currently available microscopic methods, conventional optical microscopy remains the most widespread imaging technique because it is noninvasive, nonionizing, reliable, inexpensive, and easy to use. Two of the most extensively used and promising modern optical imaging techniques are confocal microscopy and optical coherence tomography (OCT). Because of their ability for high–resolution, minimally-invasive optical sectioning, confocal and OCT techniques not only enable three-dimensional high-resolution microstructure imaging of bulk tissue specimens, but are also used to study cellular and intracellular structures and processes. These technologies have been applied to human brain function imaging, living cell tracing, gene mapping, breast cancer detection and high-speed intravascular monitoring. The aims of this research include studying the fundamental principles, critical parameters, advantages, and limitations of the confocal microscopy and OCT as high-sensitive three-dimensional bioimaging and sensing modalities, evaluating critical parameters of novel fiber-optic-based systems and investigating novel techniques for characterization and diagnostics of tissue optical properties (see Fig. 3).
![Diagram of: Optical arrangement (a) of a submicron reflection confocal microscope with optical fiber output; experimental (open circles) and analytical (full circles) axial confocal responses (b); typical confocal image of neuron growth in mouse brain [Science, 300 (2003) 76] (c).](opdt-fig3a.gif) |
![Diagram of: Optical arrangement (a) of a submicron reflection confocal microscope with optical fiber output; experimental (open circles) and analytical (full circles) axial confocal responses (b); typical confocal image of neuron growth in mouse brain [Science, 300 (2003) 76] (c).](opdt-fig3b.gif) |
![Diagram of: Optical arrangement (a) of a submicron reflection confocal microscope with optical fiber output; experimental (open circles) and analytical (full circles) axial confocal responses (b); typical confocal image of neuron growth in mouse brain [Science, 300 (2003) 76] (c).](opdt-fig3c.gif) |
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| Figure 3. Optical arrangement (a) of a submicron reflection confocal microscope with optical fiber output; experimental (open circles) and analytical (full circles) axial confocal responses (b); typical confocal image of neuron growth in mouse brain [Science, 300 (2003) 76] (c). | ||
Publications
Optical spectroscopy techniques have shown great promise for use in minimally invasive detection of precancerous lesions. Several areas of research are being pursued to elucidate the fundamental working mechanisms involved with optical diagnostic devices and their use in a safe and effective manner:
Determination of Optical Properties Using Reflectance Spectroscopy
For optical diagnostic devices, it is important that optical property data be
collected in vivo, as the literature indicates that in vitro
data does not accurately represent the case for living tissue. At present, there
is minimal in vivo data on the optical properties of internal epithelial
tissues in the ultraviolet A to visible wavelength ranges (320-700 nm) –
the regime most relevant to in vivo optical diagnostic techniques (eg.
reflectance and fluorescence spectroscopy). The objective of this work is to
improve the accuracy of our combined experimental/computational/analytical approach
for determining in vivo tissue optical properties using reflectance spectroscopy.
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| Figure 2. Example of a fiberoptic probe used in our early studies . | Figure 3. Graph of results (true values vs. estimated values). |
Device-Tissue Interface Design For Fluorescence Spectroscopy
Fluorescence-based diagnostic devices have incorporated various approaches for
delivering excitation light and collecting fluorescence, such as wide-field
imaging and a myriad of fiberoptic probe arrangements. Differences in device-tissue
interface were assumed to have negligible effect on detected signals. Our recent
work indicates that illumination-collection parameters can play a role in determining
the spatial origin of detected fluorescence. Fluorescence imaging of epithelial
tissue sections has shown that individual layers may contain diagnostically
relevant information. As a result, there has recently been increasing interest
in optimizing probe design for specific applications. One of our current objectives
is to characterize the effect of oblique-incidence illumination-collection design
(Figure 4) on the origin of detected signals (Figure 5).
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| Figure 4. Effect of oblique-incidence illumination-collection design. | Figure 5. Effect of design on the origin of detected signals. |
Fluorescence From Intravenous Medications
The strength and spectral distribution of in vivo fluorescence signals
used for medical diagnostics depends on the species present and the molecular
environment. These signals may be altered if exogenous constituents either quench
or produce fluorescence. One report found that topical drugs can interfere with
fluorescence signals from tissue (Agrawal et al., 1999, Lasers Surg Med, 25:237-249.).
Presently, clinicians exclude potential patients from fluorescence studies who
have been treated with photodynamic therapies and therapeutic retinoids for
about 90 days post-treatment. However, fluorescence resulting from a photodynamic
therapy agent has been shown to persist in esophageal tissues much longer than
90 days (Pfefer et al, 2001, Photochem Photobiol 73:664-668). Using a high-sensitivity
spectrofluorometer, we are measuring the absorption and fluorescence characteristics
of common medications (such as Ciproflaxacin, Figure 6) which have the potential
to interfere with optical diagnostic devices.
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| Figure 6. High-sensitivity spectrofluorometer. | Figure 7. Flourescence emission spectrum of ciprofloxacin. |
These projects are being performed in collaboration with top academic research groups at Rice University (Houston, Texas), the University of Arizona and the Beckman Laser Institute and Medical Clinic at the University of California at Irvine. A significant portion of our lab’s research has been performed by highly talented undergraduate and graduate-level student researchers from Johns Hopkins University, Catholic University of America and Marquette University.
Publications
Reprint requests: tdp@cdrh.fda.gov
Therapeutic lasers are used in a number of medical specialties. Today, dermatologic indications are one of the hottest areas (no pun intended) in laser medicine. On a daily basis, laser advertisements for corrective corneal eye surgery (LASIK) are broadcast. In oncology, laser light is combined with a photo-sensitizer (drug) and used to kill cancer cells, or in ophthalmology to restore eyesight. These therapeutic applications of laser light have brought dramatic improvements in patient outcomes. Indeed, most of these indications were not clinically available ten years ago.
The therapeutic laser program at OSEL develops techniques for evaluating the performance of these laser devices. Because of the wide variety of diversified medical indications, this presents both scientifically challenging and very stimulating environment. Other the past few years, the following topics have been investigated; the transmission properties of Argon laser radiation in blood, the appearance of bubbles during laser angioplasty, the surgical performance of sapphire optical tips during Nd:YAG laser surgery, the optical performance of side-firing optical fibers that are used in urologic surgery, and the optical properties of Intralipid, a nutritional supplement that is commonly used in optical research as a light scattering medium. Most recently, the relationship of the mechanical strength of ablative phantoms to their ablation efficiency has been investigated. In the very near future, the relationship between corneal hydration and the ablation efficiency of an Er:YAG laser will be explored. These research topics have resulted in collaborations with the following institutions: Oregon Medical Laser Center, University of Texas Biomedical Engineering Program, M.D. Anderson Cancer Center, and the Walter Reed Hospital Cardiology Department.
The therapeutic laser laboratory currently has the following lasers, a Ho:YAG laser, a pulsed Nd:YAG laser with harmonic output capability, and a high power Argon Ion laser. Other laser sources in the Division of Physics are also available.
Updated January 27, 2005
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