Instrument equivalence and quality control are critical elements of multi-center clinical trials. We currently have five identical Diffuse Optical Spectroscopic Imaging (DOSI) instruments enrolled in the American College of Radiology Imaging Network (ACRIN, #6691) trial located at five academic clinical research sites in the US. The goal of the study is to predict the response of breast tumors to neoadjuvant chemotherapy in 60 patients. In order to reliably compare DOSI measurements across different instruments, operators and sites, we must be confident that the data quality is comparable. We require objective and reliable methods for identifying, correcting, and rejecting low quality data. To achieve this goal, we developed and tested an automated quality control algorithm that rejects data points below the instrument noise floor, improves tissue optical property recovery, and outputs a detailed data quality report. Using a new protocol for obtaining dark-noise data, we applied the algorithm to ACRIN patient data and successfully improved the quality of recovered physiological data in some cases.
We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.
Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon
microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in
vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly
presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have
developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the
disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have
been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as
deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue
imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more
shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in
overlapping tissue regions) between the different technologies.
Scanning laser image correlation (SLIC) is an optical correlation technique for measuring the fluid velocity of particles suspended in a liquid. This technique combines laser scanning of an arbitrary pattern with pair cross-correlation between any two points in the pattern. SLIC overcomes many of the limitations of other optical correlation techniques for flow measurement, such as laser speckle, spatial temporal image correlation spectroscopy, and two-foci methods. One of the main advantages of SLIC is that the concept can be applied to measurements on a range of scales through simple zooming or modifications in the instrumentation. Additionally, SLIC is relatively insensitive to instrument noise through the use of correlation analysis and is insensitive to background. SLIC can provide detailed information about the direction and pattern of flow. SLIC has potential applications ranging from microfluidics to blood flow measurements.
Ongoing multi-center clinical trials are crucial for Biophotonics to gain acceptance in medical imaging. In these trials,
quality control (QC) and assurance (QA) are key to success and provide "data insurance". Quality control and assurance
deal with standardization, validation, and compliance of procedures, materials and instrumentation. Specifically, QC/QA
involves systematic assessment of testing materials, instrumentation performance, standard operating procedures, data
logging, analysis, and reporting. QC and QA are important for FDA accreditation and acceptance by the clinical
community. Our Biophotonics research in the Network for Translational Research in Optical Imaging (NTROI) program
for breast cancer characterization focuses on QA/QC issues primarily related to the broadband Diffuse Optical
Spectroscopy and Imaging (DOS/I) instrumentation, because this is an emerging technology with limited standardized
QC/QA in place. In the multi-center trial environment, we implement QA/QC procedures: 1. Standardize and validate
calibration standards and procedures. (DOS/I technology requires both frequency domain and spectral calibration
procedures using tissue simulating phantoms and reflectance standards, respectively.) 2. Standardize and validate data
acquisition, processing and visualization (optimize instrument software-EZDOS; centralize data processing) 3.
Monitor, catalog and maintain instrument performance (document performance; modularize maintenance; integrate new
technology) 4. Standardize and coordinate trial data entry (from individual sites) into centralized database 5. Monitor,
audit and communicate all research procedures (database, teleconferences, training sessions) between participants
ensuring "calibration". This manuscript describes our ongoing efforts, successes and challenges implementing these
strategies.
Accurate blood flow measurements during surgery can improve an operation's chance of success. We developed near-infrared spatio-temporal image spectroscopy (NIR-STICS), which has the potential to make blood flow measurements that are difficult to accomplish with existing methods. Specifically, we propose the technique and we show feasibility on phantom measurements. NIR-STICS has the potential of measuring the fluid velocity in small blood vessels (less than 1 mm in diameter) and of creating a map of blood flow rates over an area of approximately 1 cm2. NIR-STICS employs near-infrared spectroscopy to probe inside blood vessel walls and spatiotemporal image correlation spectroscopy to directly—without the use of a model—extract fluid velocity from the fluctuations within an image. We present computer simulations and experiments on a phantom system that demonstrate the effectiveness of NIR-STICS.
Using a broadband spectral technique, we recently showed [J. Biomed. Opt. 10, 064009 (2005)] that during visual stimulation of the cat brain there were not only changes in oxy- and deoxyhemoglobin levels, reminiscent of the optical blood oxygenation level dependence (BOLD) effect reported in humans, but also the apparent water content of the tissue and the optical scattering contribution decreased during stimulation. These relatively fast changes (in seconds) in water tissue content are difficult to explain in physiological terms. We developed a simple model to explain how local vasodilation, which occurs as a result of the stimulation, could cause this apparent change in water content. We show that in a phantom model we can obtain spectral effects similar to those observed in the cat brain such as the apparent decrease of the water spectral component without changing the water content of the bath in which the phantom measurements were performed. Furthermore, using the phantom model, we show that the relative apparent changes in the spectral components due to vasodilation during stimulation are roughly comparable in magnitude to the changes in tissue chromophores due to the optical equivalent of the BOLD effect reported in the literature.
We developed a spectral technique that is independent of the light transport modality (diffusive or nondiffusive) to separate optical changes in scattering and absorption in the cat's brain due to the hemodynamic signal following visual stimulation. We observe changes in oxyhemoglobin and deoxyhemoglobin concentration signals during visual stimulation reminiscent of the functional magnetic resonance imaging (fMRI) blood oxygenation level dependence (BOLD) effect. Repeated measurements at different locations show that the observed changes are local rather than global. We also determine that there is an apparent large decrease in the water concentration and scattering coefficient during stimulation. We model the apparent change in water concentration on the separation of the optical signal from two tissue compartments. One opaque compartment is featureless (black), due to relatively large blood vessels. The other compartment is the rest of the tissue. When blood flow increases due to stimulation, the opaque compartment increases in volume, resulting in an overall decrease of tissue transmission. This increase in baseline absorption changes the apparent relative proportion of all tissue components. However, due to physiological effects, the deoxyhemoglobin is exchanged with oxyhemoglobin resulting in an overall increase in the oxyhemoglobin signal, which is the only component that shows an apparent increase during stimulation.
Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 µm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.
We present a re-engineered frequency-domain tissue oximeter operating in the near-infrared spectral region. This instrument is based on the multi-distance measurement protocol, which we have implemented in our original design by multiplexing multiple light sources. The new instrument uses intensity modulated (110 MHz) laser diodes emitting at 750 and 840 nm. The laser diodes are coupled to glass optical fibers (600 micrometer core diameter). The average light intensity delivered to the tissue is about 3 mW. The multiplexing electronics are based on solid state switches that allow for acquisition times per point as short as tens of milliseconds. Our tests on phantoms and in vivo with the new oximeter have shown significant improvement in terms of stability, reliability, and reproducibility with respect to the original prototype. Furthermore, by using optical fibers we achieve a high versatility in the design of the measuring probe, permitting custom design for various tissue contours and different measurements. To verify the improved performance of the new oximeter, we have performed an in vivo test consisting of monitoring the hemoglobin saturation (Y) and concentration (THC) on the calf of 18 healthy volunteers during walking and running routines.
In the near-infrared spectral region (700 - 900 nm) light penetrates a few centimeters into tissues and hemoglobin dominates the absorption. Consequently, in vivo near-infrared tissue absorption spectroscopy becomes difficult for endogenous compounds of biological interest other than hemoglobin. Exogenous chromophore detection by fluorescence spectroscopy has the potential to provide enhanced sensitivity and specificity for in vivo optical tissue spectroscopy, facilitating the study of many important metabolites in tissues other than hemoglobin. We have performed measurements of the dc fluorescence intensity generated by a fluorophore (rhodamine B) homogeneously dissolved inside a highly scattering tissue-simulating phantom (aqueous suspension of titanium-dioxide particles). The phantom was prepared with optical coefficients (absorption and reduced scattering) similar to those of tissue in the near-infrared; these coefficients were measured with a frequency-domain spectrometer. Measurable dc fluorescence intensity signals from 1 nM rhodamine concentrations inside the phantom are reported. Furthermore, we were able to resolve changes in rhodamine concentration on the order of 1% using the dc fluorescence intensity. This dc fluorescence sensitivity is characterized experimentally at two concentrations (55 and 360 nM) and over a range of source-detector separations. Other aspects of the sensitivity are discussed over a large range of concentrations using a fluorescence photon migration model.
Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams should be considered when studying physiology and metabolism of vital cells.
We report on power thresholds for cell damage in femtosecond two-photon NIR microscopes and discuss interactions between vital cells and high intense femtosecond kW pulses in the TW/cm2 (1012 W/cm2) range. The influence of femtosecond NIR pulses on cell metabolism and cell vitality was studied by autofluorescence imaging, morphology studies, employment of vitality kits and sensitive cell cloning assays. We show that cells remain unaffected by high intense NIR femtosecond pulses below certain power thresholds. This allows nondestructive nonlinear 3D fluorescence imaging of vital cells. Above these thresholds, giant cell growth, failed cell division, complete cell destruction, and intracellular plasma formation occur.
We report the first two-photon excited autofluorescence measurements in single cells induced by continuous wave NIR laser microbeams. In particular we demonstrate NIR-excited NAD(P)H fluorescence of motile spermatozoa in a single-beam gradient force optical trap ('optical tweezers'). In addition, two-photon NIR excited autofluorescence imaging and 'tau- mapping' of CHO cells was performed with laser scanning microradiation of a tunable femtosecond-Ti:sapphire laser. Cellular response on autofluorescence to two-photon absorption of NIR radiation was compared with exposure to UVA light (one-photon absorption).
Our research is aimed at the development of a frequency-domain instrument for conducting non-invasive, real-time, near-infrared, optical tomography of tissue in vivo. Our goal is to reconstruct a spatial map of the optical properties of a strongly scattering medium in a semi-infinite-geometry sampling configuration. Specifically, we focus our attention on the absorption coefficient ((mu) a) and the reduced scattering coefficient ((mu) s') of the medium. We have developed a frequency- domain measurement protocol (which we call precalibrated), which permits one to recover the values of (mu) a and (mu) s' of a uniform tissue-like phantom from a measurement at a single source-detector separation and a single modulation frequency. It requires a preliminary reference measurement on a calibration sample of known optical properties before the measurement on the investigated sample. This approach is in principle rigorous only in macroscopically homogeneous media. We have verified that the equations valid for uniform media can still be applied to yield qualitative information on the optical nature of the inhomogeneity if the effect of macroscopic inhomogeneities on the measured phase and intensity is not too large. In vitro measurements on turbid media containing scattering and absorbing homogeneities, with optical properties very similar to the background medium, gave encouraging results. We plan to implement this measurement protocol in a multisource, multidetector instrument for optical tomography.
Near infrared optical imaging is emerging as a potentially important imaging modality, because it offers real time data access, portability, cost-effectiveness, and the relatively safe use of non-ionizing radiation. Reconstruction of images by optical tomography is complicated by the diffusive character of light propagation in optically heterogeneous tissue. The spatial volume element probed by the light path between the light source and optical detector is rather wide and depends on a variety of experimental and instrumental factors. We have published an optical image of the hand in air based on photon density wave distribution characteristics, using both steady-state (intensity) and frequency-domain (phase and modulation) experimental conditions. Since then, we have developed new instrumentation, better measurement protocols, some reconstruction algorithms and a more complete theoretical understanding of photon diffusion in both homogeneous and heterogeneous media. We have now performed frequency-domain measurements (at a modulation frequency of 160 MHz with 760 nm near infrared light) with the hand immersed in a scattering fluid (the infinite geometry arrangement). The advantages of our current approach include the spectroscopic resolution of physiologically interesting tissue regions, greater spatial resolution, the generation of absorption and reduced scattering coefficient maps of the image, rapid data acquisition, real time simultaneous display of the experimental parameters and calculated optical parameters and the possibility of obtaining some tomographic reconstruction.
In this paper, we present a series of measurements made with a portable frequency-domain near-infrared tissue spectrometer (OMNIA). This is the first application of the OMNIA in a clinical setting. All of the measurements presented here were taken in vivo, most were on human subjects. We report the results of three experiments: (1) A simple ischemia/plethysmography experiment, which indicates ability of the instrument to noninvasively, continuously monitor the hemoglobin saturation of a limb. (2) A survey of hemoglobin saturation in patients with peripheral vascular disease. (3) An animal experiment to demonstrate the correlation of our instrument readings with results from established techniques for measuring hemoglobin saturation. We measured the absorption and reduced scattering coefficients of the tissue at two wavelengths (715 nm and 850 nm). From the absorption coefficients, we calculated the concentrations of oxygenated and deoxygenated hemoglobin ([HbO2] and [Hb]), which immediately yield the hemoglobin saturation (Y) and the total blood volume (T) in the tissue. Our preliminary results indicate some of the potential of the instrument and the areas for future improvement of it.
The emergence of time-resolved fluorescence techniques has been very important to the study of biochemical, biophysical and biomedical research areas. Many of the advances in these fields have been technology driven. The Laboratory for Fluorescence Dynamics has been actively involved in development of frequency domain technology for a variety of applications. Our recent work on microwave super heterodyning detectors, fluorescence lifetime resolved spectroscopy, frequency domain fluorescence imaging microscopy, fluorescence lifetime resolved stopped-flow kinetics and global methods of data analysis is summarized in this report. We provide examples of how these new technologies are applied.
Frequency domain spectroscopy provides a quantitative measure of the optical properties, namely (mu) a and (mu) s' coefficient spectra, of multiply scattering, macroscopically homogeneous media or tissue. The diffusion model for photon transport provides the theoretical framework for the analytical expressions of the optical properties. Experimentally, we intensity modulated (60 MHz) a light emitting diode, which emits between 620 - 700 nm. From data sets of relative phase shifts and average intensity at different source- detector separations, we calculated on the basis of the analytical expressions a wavelength resolved absorption (mu) a ((lambda) ) and scattering (mu) s' ((lambda) ) coefficient spectrum. The test material was methylene blue, whose absorption spectrum (maximum 656 nm) closely matches the wavelength profile of the diode source. The multiply scattering, macroscopically homogeneous medium for dissolving the methylene blue was provided by a diluted fat emulsion, Liposyn III. The concentrations of both the absorbing and scattering materials were adjusted to correspond to ranges typical of (mu) a and (mu) s' in tissues. We obtained quantitative agreement between the measured (mu) a ((lambda) ) in the scattering medium and a control solution measured in a spectrophotometer under non-scattering conditions.
High frequency, intensity-modulated light waves are attenuated and phase-shifted by the absorption and scattering properties of highly scattering media, such as tissue. The simultaneous measurement of the average light intensity, modulation amplitude, and phase- shift at a fixed distance from a sinusoidally modulated light source, permits a quantitative determination of the absolute values of the absorption and scattering coefficients from a frequency-domain scan. Our studies have established the range of modulation frequencies that give the highest sensitivity to changes of the optical parameters in model systems. We have measured the optical absorption spectra of dyes suspended in highly scattering media. These spectra match those found in non-scattering media. This frequency-domain approach provides a simple method to perform quantitative spectroscopy in highly scattering media.
Our laboratory has developed a modular laser tomography system, with pulsed or amplitude modulated (MHz to GHz), near infrared lasers that deliver a probing beam to the tissue of interest through a fiber optic. After the incoming light is scattered and attenuated by the tissue, a detector or imaging fiber optic bundle delivers it to a point detector (photomultiplier tube) which is heterodyned with the modulation frequency to yield the phase delay and demodulation resulting from the light-tissue interaction. The CCD electronics are phase-locked with those of the digitizer to minimize pixel jitter and, in addition, an external clock synchronizes the detector units with the modulated laser source. The digitized time slices are integrated into four bins corresponding to four quadrants of the cross correlation period. The final processing step, a fast Fourier transform, generates the phase shift, demodulation, and average intensity data suitable for image reconstruction.
Light propagation in turbid media can be described by photon diffusion. In the frequency domain, sinusoidally intensity-modulated light gives rise to diffusive waves which have a coherent front. In a homogeneous medium, the wave front propagates with a constant phase velocity and the amplitude attenuates exponentially as the diffusional wave advances. We have studied the diffusion approximation to the one-speed linear transport equation with a sinusoidally intensity modulated point source of particles and performed experiments using frequency domain detection methods on homogeneous scattering and absorbing media to test the applicability of the above mentioned transport equation to photon migration in turbid media. We have used the analytical solutions of the linear transport equation in homogeneous, infinite media to determine via a simple analysis of our frequency domain data the linear scattering and absorption coefficients.
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