Paul M. Kasili

Development of an advanced hyperspectral imaging (HSI) system with applications for cancer detection.

An advanced hyper-spectral imaging (HSI) system has been developed having obvious applications for cancer detection. This HSI system is based on state-of-the-art liquid crystal tunable filter technology coupled to an endoscope. The goal of this unique HSI technology being developed is to obtain spatially resolved images of the slight differences in luminescent properties of malignant versus non-malignant tissues. In this report, the development of the instrument is discussed and the capability of the instrument is demonstrated by observing mouse carcinomas in-vivo. It is shown that the instrument successfully distinguishes between normal and malignant mouse skin. It is hoped that the results of this study will lead to advances in the optical diagnosis of cancer in humans.

Nanosensor for in vivo measurement of the carcinogen benzo[a]pyrene in a single cell.

This work describes the fabrication and the application of an antibody-based fiber-optic nanosensor for in situ measurements of the carcinogen benzo[a]pyrene (BaP) in a single cell. This antibody-based spectroscopic nanosensor is miniaturized enabling the detection of fluorescent analytes in single cells. In addition to measuring fluorescent analytes in single cells, the nanosensor has the potential to be applied for both diagnostic and proteomics purposes. In this work, the human breast carcinoma cell line, MCF-7, was used as the model system to perform BaP measurements in single cells. A standard concentration curve for BaP was established and used to perform quantitative analyses of BaP in individual cells. From these analyses, it was estimated that the concentration of BaP in the individual cells investigated was approximately 3.61 x 10(-10) M. The results obtained demonstrate the application of antibody-based nanosensors for performing in situ measurements inside a single cell.

Development of a multi-spectral imaging system for medical applications

We describe the development of a multi-spectral imaging (MSI) system based on a rapid-scanning solid-state device, an acousto-optic tunable filter (AOTF), for wavelength selection and a two-dimensional charge-coupled device for detection. The MSI device is designed for in vivo optical detection in medical diagnostic applications. Unlike conventional grating spectrometers, the AOTF is a miniature solid-state device that has no moving parts, and can be rapidly tuned to any wavelength within its operating range. The large aperture of the AOTF and its high spatial resolution allows the optical image from an imaging fibre optic probe to be recorded by the detector. These characteristics, combined with their small size, make AOTFs important new alternatives to conventional monochromators, especially for spectral imaging in biomedical applications. The MSI can also be used for dual-modality diagnostics to detect both fluorescence and diffuse reflectance images. The usefulness and potential of the MSI system is illustrated in several applications of biomedical interest, such as reflectance fluorescence imaging of skin and brain tissues.

Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications

A major advantage of flow cytometry is its flexible and open instrument configuration, which is highly suitable for systems integration. This flexibility permits the coupling of auxiliary instrumentation that may offer the measurement of parameters other than those typically measured by this multiparameter measurement technique. On the basis of this advantage, we explore the principle and application of hyperspectral imaging (HSI), which has the potential to be a useful add‐on feature to flow cytometry applications. Application of HSI to flow cytometry involves the acquisition of spatial information and rendering it in spectral form. In this work, we describe the development and application of an HSI system which provides both spectral and spatial information. Spectral information was generated by obtaining an entire spectrum of a single sample site within a wavelength region of interest, while spatial information was generated by recording a two‐dimensional (2D) image of an area of the sample of interest at one specific wavelength. HSI is a promising additional feature to flow cytometry since it can provide both spatial (image format) and spectral information in addition to the multiparameter information already available from flow cytometry measurements.

Liposome encapsulated gold nanoshells for NanoPhototherapy induced hyperthermia

Gold nanoshells have been extensively studied since their invention and their role in both in vitro and in vivo photothermal therapy has been explored and demonstrated. In this work, we investigate liposomal delivery of gold nanoshells and evaluate their effects for in vitro NanoPhototherapy induced hyperthermia in human mammary carcinoma cells. In addition, we compare the application of liposome encapsulated gold nanoshells and free standing gold nanoshells in NanoPhototherapy. NanoPhotoTherapy induced hyperthermia was performed using a 785 nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, we monitored apoptosis by detecting capase-9 activity.

Development of a Fluorescence Detection System Using Optical Parametric Oscillator (OPO) Laser Excitation for in Vivo Diagnosis

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. in vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

Photothermal Treatment of Human Carcinoma Cells Using Liposome-Encapsulated Gold Nanoshells

We report the application of liposome-encapsulated gold nanoshells for in vitro photo-induced hyperthermia in human mammary carcinoma cells. In addition to evaluating their effects in vitro, we compared the application liposome-encapsulated gold nanoshells and free-standing gold nanoshells for NanoPhotoTherapy (NPT). NPT-induced hyperthermia was performed using a 785-nm near-infrared light from a diode laser and the in vitro effects were evaluated using nucleic acid molecular probes by fluorescence microscopy. Additionally, we monitored the effectiveness of NPT by detecting apoptosis via capase-9 activity.

DETECTION OF POLYCYCLIC AROMATIC COMPOUNDS IN SINGLE LIVING CELLS USING OPTICAL NANOPROBES

Exposure of mammalian cells to polycyclic aromatic compounds (PACs) such as the carcinogen benzo[a]pyrene (BaP) leads to the formation of DNA adducts N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) with adenine and guanine nucleotides, which are integral parts of DNA, RNA, and ATP. DNA adduct formation causes alteration of the DNA (RNA) sequence since neither adenine nor guanine can normally bind to its complementary nucleotide base, thymine (uracil) and cytosine respectively. The inability to form these bonds leads to mutations in the DNA double-helix structure during DNA replication, and eventually carcinogenesis. Therefore, the capability to detect and measure PAC species such as BaP in single living cells is important for studies required to establish the limits of BaP exposure necessary for carcinogenesis. Along these lines, we have developed antibody-based optical nanoprobes capable of detecting and measuring BaP in single living cells. The results obtained in this work demonstrate the practical application of antibody-based nanoprobes for performing measurements inside single living cells with their elements and their relationships intact.

Application of ultrasonic techniques for brain injury diagnosis

In this work, we evaluate methods for detecting brain injury using ultrasound. We have used simulations of ultrasonic fields in the head to model the phase distortion of the skull. In addition we present experimental data from the crania of large animals. The experimental data help us understand and evaluate the performance of different transducers in acquiring the backscatter data from the brain through the skull. Both the simulations and acquired data illustrate the superiority of lower-frequency (<EQ 1 MHz) ultrasonic fields for transcranial acquisition of signals from inside the brain. Additionally, the experimental work shows that the higher-frequency (5 MHz) ultrasound can also be useful in acquiring clean nearfield data to help detect the position of the inner boundary of the skull.

Hyperspectral imaging using AOTF and NIR sensing of buried objects and landmines

The detection of landmines and buried objects requires methods that can cover large areas rapidly while providing the required sensitivity to detect the optical and spectroscopic contrasts in soil properties that can reveal their presence. These conditions on contrast and coverage can be met by capturing images of the soil at wavelengths which are sensitive to the properties modified by the presence of buried objects. In this work we investigate both imaging and scanning methods which may have some utility for the detection problem. In the imaging approach, we capture hyperspectral reflection images using an acousto-optic tunable filter (AOTF) and fluorescence images using a long-pass filter. For the scanning method, we acquire data point-by-point over a two-dimensional grid with a single emitter/detector pair. The results illustrate the potential of these two approaches for detection of landmines and buried objects.