Diffuse reflectance and fluorescence spectroscopy are used to detect histopathological abnormalities of an epileptic brain in a human subject study. Static diffuse reflectance and fluorescence spectra are acquired from normal and epileptic brain areas, defined by electrocorticography (ECoG), from pediatric patients undergoing epilepsy surgery. Biopsy specimens are taken from the investigated sites within an abnormal brain. Spectral analysis reveals significant differences in diffuse reflectance spectra and the ratio of fluorescence and diffuse reflectance spectra from normal and epileptic brain areas defined by ECoG and histology. Using these spectral differences, tissue classification models with accuracy above 80% are developed based on linear discriminant analysis. The differences between the diffuse reflectance spectra from the normal and epileptic brain areas observed in this study are attributed to alterations in the static hemodynamic characteristics of an epileptic brain, suggesting a unique association between the histopathological and the hemodynamic abnormalities in an epileptic brain.
In this pilot study, we explored the potential of using a diffuse reflectance imaging system to extract interictal
pathophysiological characteristics of epileptic cortex in an intraoperative setting. The imaging system was able to
simultaneously measure diffuse reflectance signals at two distinct wavelengths (500 and 700 nm) from the entire
exposed cortical surface. It was used to study ten pediatric patients during their epilepsy surgery. Diffuse reflectance
images, Rd(x,y,λ,t) at 500 nm and 700 nm, were acquired at a 5 Hz rate for at least 200 seconds. Post imaging analysis
identified a unique local frequency oscillation (LFO), below respiration rate, existed in Rd(x,y,500 nm,t) and Rd(x,y,700
nm,t). Mapping the spectral densities of LFOs over the cortical surface identified the spatial distribution of the LFOs. In
almost all ten patients studied, the location demonstrating strong LFOs coincided with the epileptic cortex determined
using ECoG. However, some LFOs were found in close proximity to functional areas according to fMRI. We further
used the correlation coefficient map to identify those pixels with similar waveforms for better demarcation. These
preliminary results support the feasibility of using wavelength-dependent diffuse reflectance imaging to intra-operatively
detect epileptic cortex.
The concept of using diffuse reflectance spectroscopy to distinguish intraoperatively between pediatric brain tumors and normal brain parenchyma at the edge of resection cavities is evaluated using an in vivo human study. Diffuse reflectance spectra are acquired from normal and tumorous brain areas of 12 pediatric patients during their tumor resection procedures, using a spectroscopic system with a handheld optical probe. A total of 400 spectra are acquired at the rate of 33 Hz from a single investigated site, from which the mean spectrum and the standard deviation are calculated. The mean diffuse reflectance spectra collected are divided into the normal and the tumorous categories in accordance with their corresponding results of histological analysis. Statistical methods are used to identify those spectral features that effectively separated the two tissue categories, and to quantify the spectral variations induced by the motion of the handheld probe during a single spectral acquisition procedure. The results show that diffuse reflectance spectral intensities between 600 and 800 nm are effective in terms of differentiating normal cortex from brain tumors. Furthermore, probe movements induce large variations in spectral intensities (i.e., larger standard deviation) between 400 and 600 nm.
We explore the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at different developing stages. An animal study is conducted using rats with surgically induced myocaridal infarction (MI). In vivo fluorescence spectra at 337-nm excitation and diffuse reflectance between 400 and 900 nm are measured from the heart. Spectral acquisition is performed: 1. for normal heart tissue; 2. for the area immediately surrounding the infarct; and 3. for the infarcted tissue itself, one, two, three, and four weeks into MI development. Histological and statistical analyses are used to identify unique pathohistological features and spectral alterations associated with the investigated regions. The main alterations (p<0.05) in diffuse reflectance spectra are identified primarily between 450 and 600 nm. The dominant fluorescence alterations are increases in peak fluorescence intensity at 400 and 460 nm. The extent of these spectral alterations is related to the duration of the infarction. The findings of this study support the concept that optical spectroscopy could be useful as a tool to noninvasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing real-time feedback to surgeons during various surgical interventions for MI.
KEYWORDS: Brain, Tissue optics, Diffuse reflectance spectroscopy, Epilepsy, Data analysis, Tissues, Tumors, Control systems, Statistical analysis, In vivo imaging
This research investigated the feasibility of using time-dependent diffuse reflectance spectroscopy to differentiate
pediatric epileptic brain tissue from normal brain tissue. The optical spectroscopic technique monitored the dynamic
optical properties of the cerebral cortex that are associated with its physiological, morphological, and compositional
characteristics. Due to the transient irregular epileptic discharge activity within the epileptic brain tissue it was
hypothesized that the lesion would express abnormal dynamic optical behavior that would alter normal dynamic
behavior. Thirteen pediatric epilepsy patients and seven pediatric brain tumor patients (normal controls) were recruited
for this clinical study. Dynamic optical properties were obtained from the cortical surface intraoperatively using a timedependent
diffuse reflectance spectroscopy system. This system consisted of a fiber-optic probe, a tungsten-halogen light
source, and a spectrophotometer. It acquired diffuse reflectance spectra with a spectral range of 204 nm to 932 nm at a
rate of 33 spectra per second for approximately 12 seconds. Biopsy samples were taken from electrophysiologically
abnormal cortex and evaluated by a neuropathologist, which served as a gold standard for lesion classification. For data
analysis, spectral intensity changes of diffuse reflectance in the time domain at two different wavelengths from each
investigated site were compared. Negative correlation segment, defined by the periods where the intensity changes at the
two wavelengths were opposite in their slope polarity, were extracted. The total duration of negative correlation, referred
to as the "negative correlation time index", was calculated by integrating the negative correlation segments. The negative
correlation time indices from all investigated sites were sub-grouped according to the corresponding histological
classifications. The difference between the mean indices of two subgroups was evaluated by standard t-test. These
comparison and calculation procedures were carried out for all possible wavelength combinations between 400 nm and
800 nm with 2 nm increments. The positive group consisted of seven pathologically abnormal test sites, and the negative
group consisted of 13 normal test sites from non-epileptic tumor patients. A standard t-test showed significant difference
between negative correlation time indices from the two groups at the wavelength combinations of 700-760 nm versus
550-580 nm. An empirical discrimination algorithm based on the negative correlation time indices in this range produced
100% sensitivity and 85% specificity. Based on these results time-dependent diffuse reflectance spectroscopy with
optimized data analysis methods differentiates epileptic brain tissue from normal brain tissue adequately, therefore can
be utilized for surgical guidance, and may enhance the surgical outcome of pediatric epilepsy surgery.
The objective of this in vitro tissue study is to investigate the feasibility of using optical spectroscopy to differentiate pediatric neoplastic and epileptogenic brain from normal brain. Specimens are collected from 17 patients with brain tumors, and from 26 patients with intractable epilepsy during surgical resection of epileptogenic cerebral cortex. Fluorescence spectra are measured at excitations of 337, 360, and 440 nm; diffuse reflectance spectra are measured between 400 and 900 nm from each specimen. Pathological analysis is performed to classify abnormalities in brain specimens, and its findings are correlated with spectral data. Statistically significant differences (p<0.01) are found for both raw and normalized diffuse reflectance and fluorescence spectra between 1. neoplastic brain and normal gray matter, 2. epileptogenic brain and normal gray matter, and 3. neoplastic brain and normal white matter. However, no distinct spectral features are identified that effectively separate epileptogenic brain from normal white matter. The outcomes of the study suggest that certain unique compositional and structural characteristics of pediatric neoplastic and epileptogenic brain can be detected using optical spectroscopy in vitro.
Optical spectroscopy for in vivo tissue diagnosis is performed traditionally in a static manner; a snap shot of the tissue
biochemical and morphological characteristics is captured through the interaction between light and the tissue. This
approach does not capture the dynamic nature of a living organ, which is critical to the studies of brain disorders such as
epilepsy. Therefore, a time-dependent diffuse reflectance spectroscopy system with a fiber-optic probe was designed
and developed. The system was designed to acquire broadband diffuse reflectance spectra (240 ~ 932 nm) at an
acquisition rate of 33 Hz. The broadband spectral acquisition feature allows simultaneous monitoring of various
physiological characteristics of tissues. The utility of such a system in guiding pediatric epilepsy surgery was tested in a
pilot clinical study including 13 epilepsy patients and seven brain tumor patients. The control patients were children
undergoing suregery for brain tumors in which measurements were taken from normal brain exposed during the surgery.
Diffuse reflectance spectra were acquired for 12 seconds from various parts of the brain of the patients during surgery.
Recorded spectra were processed and analyzed in both spectral and time domains to gain insights into the dynamic
changes in, for example, hemodynamics of the investigated brain tissue. One finding from this pilot study is that
unsynchronized alterations in local blood oxygenation and local blood volume were observed in epileptogenic cortex.
These study results suggest the advantage of using a time-dependent diffuse reflectance spectroscopy system to study
epileptogenic brain in vivo.
Epileptogenic lesions and their margins are often difficult to define intraoperatively. We hypothesize that optical
spectroscopy can detect unique pathophysiological features of epileptogenic lesions in children and hence differentiate
them from normal brain. This hypothesis was tested by comparing the in vivo optical and fluorescence characteristics of
epileptogenic brain lesions (non-neoplastic) with those of normal brain. Patients were recruited from those receiving
epilepsy surgeries at Miami Children's Hospital. Using a portable spectroscopic system, optical characterization of brain
was performed intraoperatively. Fluorescence spectra were measured at 337 nm excitation, and diffuse reflectance
spectra were measured between 400 and 850 nm. To date, seven epilepsy patients have been enrolled in the study. A
couple interesting trends have been observed in the recorded optical spectra. First, sites within the resection zone, as
defined by the intracranial electroencephalogram data, often show higher diffuse reflectance signals than normal sites
do. This is especially prominent around 500 nm and between 650 and 850 nm. Secondly, several investigated sites with
abnormal electroencephalogram and/or pathology show a unique blue shift in their fluorescence spectra, which is not
seen in other cases.
Recently, optical spectroscopy has shown considerable promise to be used as a potential clinical tool for human brain
tumor detection and therapeutic guidance. Our group showed for the first time the possibility of using combined
autofluorescence and diffuse reflectance spectroscopy and established its applicability for human brain tumor
demarcation in previous in vitro and in vivo studies. We report in this paper the results of a clinical study designed to
further evaluate the efficacy of the approach for demarcation of brain tumors and tumor margins from normal brain
tissues in intra-operative clinical setting. Using a portable system, optical spectra were collected from the brain of 110
patients undergoing craniotomy at the Vanderbilt University Medical Center. Spectral measurements were taken from
multiple sites of tumor core, tumor margin and normal areas of brain tissues and the resulting spectra were correlated
with the corresponding histopathologic diagnosis. Using histology as the gold standard, a probabilistic multi-class
diagnostic algorithm was developed to simultaneously distinguish tumor core and tumor margin from normal brain tissue
sites using independent training and validation sets of data. An unbiased estimate of the accuracy of the model indicates
that combined autofluorescence and diffuse reflectance spectroscopy was able to distinguish tumor core and tumor
margin from normal brain tissues with an average predictive accuracy of ~88%.
OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence.
METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 ± 6, 85 ± 6, 53 ± 9 and 0 ± 3. The relative fluorescence at the same time points were 100 ± 2, 57 ± 6, 47 ± 3, and 0 ± 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.
Resection is not a viable treatment option for the majority of liver cancer patients. Alternatives to resection include thermotherapies such as radio-frequency ablation; however, these therapies lack adequate intraoperative feedback regarding the degree and margins of tissue thermal damage. In this proof of principle study, we test the ability of fluorescence and diffuse reflectance spectroscopy to assess local thermal damage in vivo. Spectra were acquired in vivo from healthy canine liver tissue undergoing radio-frequency ablation using a portable fiber-optic-based spectroscopic system. The major observed spectral alterations on thermal coagulation were a red shift in the fluorescence emission peak at 480 nm, a decrease in the overall fluorescence intensity, and an increase in the diffuse reflectance from 450 to 750 nm. Spectral changes were quantified and correlated to tissue histology. We found a good correlation between the proposed spectral correlates of thermal damage and histology. The results of this study suggest that fluorescence and diffuse reflectance spectroscopy show strong potential as tools to monitor liver tissue thermal damage intraoperatively.
Thermotherapies such as radio-frequency ablation achieve necrosis of liver tumors through thermal coagulation and are frequently employed when surgical resection is impossible. Currently, thermotherapy procedures suffer from the lack of an adequate feedback control system, making it difficult to know precisely when to terminate therapy. In vitro and in vivo studies were performed on canine liver tissue to determine the feasibility of using fluorescence and diffuse reflectance spectroscopy to provide an objective endpoint for these procedures. The fluorescence and diffuse reflectance spectra of liver tissue exhibited consistent changes over the coagulation process. In vitro results showed a shift in the primary fluorescence peak from 490 nm in the native state to 510 nm in the fully coagulated state; in addition, a three- to four-fold increase in the absolute intensity of the diffuse reflectance spectra was observed upon complete coagulation. Similar spectral alterations were obtained in vivo. Based on our results, fluorescence and diffuse reflectance spectroscopy provide a direct way to assess the biochemical and structural changes associated with tissue thermal damage; hence, they can be developed into a feedback system for thermotherapies.
Complete infiltrating brain tumor margin resection continually eludes neurosurgeons due to inherent limitations of current margin localization techniques. A need exists for an objective, on-site, real-time imaging system which can accurately localize brain tumor margins and therefore be used as a basis for image-guided surgery. Optical biopsy methods are a proven means for successful brain tissue discrimination, indicating promise for spectral imaging to fill such a need. Before testing spectral imaging for surgical guidance, various spectral imaging modalities must be systematically compared to determine the modality most conducive to the clinical setting. A liquid crystal tunable filter spectral imaging system was characterized for field of view, spatial and spectral resolution, and ability to retain spectral features acquired from a clinical single-pixel spectroscopy system. For a 35-mm diameter field of view, the system possessed a spatial resolution of 50 μm in both image dimensions and a spectral resolution which monotonically increased from 10 to 30 nm over the tuning range of the filter. Differences between imaging and single-pixel spectra for location and FWHM of fluorescence peaks from two fluorescent dye targets were summarily less than 3 nm. However, two remediable artifacts were introduced to imaging system spectra during spectral sensitivity correction.
Spectral imaging as a modality combines two powerful techniques of imaging and fluorescence spectroscopy. By generating a 2-D image of an object with fluorescence information at every pixel, spectral imaging has the potential to provide clinicians with a valuable tool which can not only diagnosis the tissue but also provide an image of the boundary of where the normal and cancerous tissue intersect. The system used in these experiments was a modified spectral imaging system from Applied Spectral Imaging (SD-200, Carlsbad, CA). The system was mounted in an off-microscope configuration so that instead of performing microscopic measurements of tissue, macroscopic measurements on the order of several millimeters in size were collected. Preliminary results indicate that the spectra acquired from human brain tissues in vitro at individual pixels of the spectral image cube appear similar to that acquired using the single pixel system. Based on the findings of this study, spectral imaging has the potential to be a useful tool for tissue diagnostics and is currently limited by the speed of data acquisition and size of the data.
An optical spectroscopy-based system for intraoperative guidance of brain tumor resection is currently under development at Vanderbilt University and Vanderbilt University Medical Center. The effectiveness of brain tumor margin detection, especially the infiltrating type, using combined in vivo autofluorescence and diffuse reflectance spectroscopy was successfully demonstrated in a preliminary clinical trial (on a total of 26 brain tumor patients). In this trial, a two-step empirical discrimination algorithm based on autofluorescence and diffuse reflectance intensity at 460 nm and 625 nm yielded 100% sensitivity and 76% specificity in differentiating infiltrating tumor margins from normal brain tissues.
A pilot in vivo study was conducted to investigate the feasibility of using optical spectroscopy for brain tumor margin detection. Fluorescence and diffuse reflectance spectra were acquired using a portable clinical spectroscopic system from normal brain tissues, tumors, and tumor margins in 21 brain tumor patients undergoing craniotomy. Results form this study show the potential of optical spectroscopy in detecting infiltrating tumor margins of primary brain tumors.
In this study, the Z-scan technique was used to examine the thermal lens effect in turbid media containing micro-spheres suspended in water. The scattering coefficients of the turbid media were varied from 10 to 100 cm-1. The experimental results suggested that the nonlinear changes in the refractive index of the scattering medium induced by high power laser heating could significantly impact the propagation of laser beam through the scattering medium. Presence of thermally-induced nonlinear changes in refractive index of tissue during high power laser irradiation may significantly alter the distribution of laser light in tissue during laser therapy.
We have studied dynamic changes in optical properties of various tissues during argon and pulse Nd:YAG laser irradiation while monitoring tissue surface temperature. High power laser radiation induces reversible and/or irreversible changes in the optical behavior of the tissue. Reversible changes in diffuse reflectance and total transmittance can be as much as 15% and 20% of initial values, respectively. We are currently investigating the mechanism responsible for the observed reversible changes.
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