SignificanceDiagnosis of cancerous and pre-cancerous oral lesions at early stages is critical for the improvement of patient care, to increase survival rates and minimize the invasiveness of tumor resection surgery. Unfortunately, oral precancerous and early-stage cancerous lesions are often difficult to distinguish from oral benign lesions with the existing diagnostic tools used during standard clinical oral examination. In consequence, early diagnosis of oral cancer can be achieved in only about 30% of patients. Therefore, clinical diagnostic technologies for fast, minimally invasive, and accurate oral cancer screening are urgently needed.AimThis study investigated the use of multispectral autofluorescence imaging endoscopy for the automated and noninvasive discrimination of cancerous and precancerous from benign oral epithelial lesions.ApproachIn vivo multispectral autofluorescence endoscopic images of clinically suspicious oral lesions were acquired from 67 patients undergoing tissue biopsy examination. The imaged lesions were classified as precancerous (n=4), cancerous (n=29), and benign (n=34) lesions based on histopathology diagnosis. Multispectral autofluorescence intensity feature maps were generated for each oral lesion and used to train and optimize support vector machine (SVM) models for automated discrimination of cancerous and precancerous from benign oral lesions.ResultsAfter a leave-one-patient-out cross-validation strategy, an optimized SVM model developed with four multispectral autofluorescence features yielded levels of sensitivity and specificity of 85% and 71%, respectively and overall accuracy of 78% in the discrimination of cancerous/precancerous versus benign oral lesions.ConclusionThis study demonstrates the potentials of a computer-assisted detection system based on multispectral autofluorescence imaging endoscopy for the early detection of cancerous and precancerous oral lesions.
Multispectral autofluorescence lifetime imaging (maFLIM) endoscopy can be used to clinically image a plurality of metabolic and biochemical autofluorescence biomarkers of oral precancer and cancer. We tested the hypothesis that maFLIM-derived autofluorescence biomarkers can be used as features in machine-learning models to automatically discriminate precancerous and cancerous from healthy oral tissue. Clinical widefield maFLIM endoscopy images of cancerous and precancerous oral lesions from 57 patients were acquired and used to develop and validate a computer-aided detection (CAD) system. This study demonstrates the potentials of a maFLIM endoscopy-based CAD system for automated in situ clinical detection of oral precancer and cancer.
Multispectral autofluorescence endoscopy is a non-invasive optical imaging modality that can provide contrast between malignant and benign oral tissue. We hypothesized that discrimination of cancerous and precancerous from benign oral lesions can be achieved through machine-learning (ML) models developed with multispectral autofluorescence intensity features. In vivo multispectral autofluorescence endoscopic images of benign, precancerous, and cancerous oral lesions were acquired from 67 patients and used to optimize ML models for discrimination between cancerous/precancerous and benign lesions. This study demonstrates the potentials of a ML-assisted system based on multispectral autofluorescence endoscopy for automated discrimination of cancerous and precancerous from benign oral lesions.
We present our 2nd generation handheld simultaneous multispectral frequency-domain FLIM endoscopic system for label-free metabolic imaging of oral cancer, with enhanced optical performance and system usability. Our custom-designed and 3D-printed handheld endoscope consists of an enclosure (6 x 3 x 3 cm3) with a rigid probe (1 cm diameter, 9 cm length) that weighs less than 125 g with all the system components, which, compared to our previous system, is significantly smaller and lighter, and has improved ergonomics and usability. The enclosure has mounts for a dual axes bi-directional MEMS scanner and a dichroic mirror, and plug-and-play ports for excitation, emission collection and rigid probe optics. The rigid probe used for oral mucosa imaging contains a three-lens imaging system that, compared to our previous system, has: an increased field of view (FOV) (6 x 6 mm2 vs. 16 x 16 mm2), improved lateral resolution (36 μm in the center and 65 μm at the edge, diffraction-limited performance across a central ± 5.5 mm field), and an extended working distance (10 mm vs. 40 mm). A 375 nm CW laser is used as the excitation source, and fluorescence emission is spectrally divided into three emission bands (405±10 nm, 440±20 nm, and 525±25 nm) targeting collagen, NADH, and FAD, which are relevant for early detection of oral cancer. Fluorescence emission is then detected by three APDs and further processed onboard an FPGA. Our clinically compatible handheld endoscope allows for noninvasive and fast in situ clinical metabolic imaging of the oral mucosa.
Fluorescence lifetime imaging (FLIM) is a technique that allows calculating the fluorescence lifetime at every pixel of an imaged fluorescent sample. The fluorescence lifetime is a property that characterizes each fluorophore and its environment, which makes FLIM a powerful quantitative analytical tool extensively used in a wide range of biomedical applications. In order to fully exploit the potentials of FLIM in the medical field, practical implementations that would enable fast and accurate in vivo FLIM imaging are needed. We present a handheld FLIM system capable of both acquiring and processing time-resolved fluorescence measurements at a pixel rate of at least 30 kHz. The handheld instrument provides a field of view of ~1 cm in diameter with an optical resolution of ~100 μm. Real-time FLIM processing is achieved by means of a bi-exponential model curve fitting algorithm based on a lookup table and pattern recognition techniques. The handheld FLIM system was validated by safely imaging fluorescence standards and the oral mucosa of healthy volunteers. The acquired fluorescence lifetime maps were in agreement with the fluorescence lifetime values estimated using the standard non-linear least square iterative reconvolution method (LSIR). These results demonstrated practical and accurate in vivo video rate FLIM imaging capabilities of this novel handheld FLIM implementation, which would facilitate practical FLIM applications, including clinical ones, such as clinical diagnosis and image guided interventions.
Increased metabolic activity, a hallmark of epithelial cell malignant transformation, induces subtle changes in the oral tissue autofluorescence. The optical “redox-ratio”, defined as the autofluorescence intensity of NADH divided by that of FAD, is sensitive to changes in the cellular metabolic rate. A decrease in the redox-ratio indicates increased cellular metabolic activity, as is typically observed in malignant cells. Specific changes in the fluorescence lifetime of both NADH and FAD have also been associated with increased metabolic activity in malignant oral epithelial cells. We therefore hypothesized that more specific biomarkers of oral cancer and dysplasia can more accurately be quantified by endogenous fluorescence lifetime imaging (FLIM). In this work, FLIM images of benign, dysplastic and early stage cancerous oral lesions from 52 patients were acquired at three emission channels (390±20nm, 452±22.5nm and >500nm) using a handheld multispectral FLIM endoscope. For each pixel, the fluorescence decays collected at the three emission bands were analyzed using a biexponential decay model, resulting on 16 FLIM-derived parameters per pixel. Statistical analysis was performed on each of the computed FLIM parameters (Wilcoxon test: Normal vs. Benign, Normal vs. Dysplasia/Cancer; Mann-Whitney test: Benign vs. Dysplasia/Cancer). Results from this analysis revealed that FLIM-derived parameters associated with collagen lifetime, NADH lifetime, FAD autofluorescence, and the optical redox ratio were statistical different between dysplastic/cancerous vs. benign oral lesions. This study provides the first demonstration for the clinical imaging of autofluorescence biochemical and metabolic biomarkers of oral epithelial cancer and dysplasia, which could potentially enable early detection of oral cancer.
A compact handheld system for simultaneous multispectral frequency-domain (FD) FLIM imaging is presented. The handheld endoscope consists of a handheld enclosure (10 X 5 X 3 cm3 in volume) with a rigid probe (0.8 cm diameter, 12 cm length). The customized enclosure holds the MEMS scanner and a dichroic mirror whose tip and tilt angle can be adjusted. The rigid probe includes four achromatic lenses (f = 30mm). Two of the four lenses form a relay system to extend the length of the probe. The most distal lens works as an objective to focus the light onto the sample. An additional lens is placed in the intermediate image plane of the relay system to increases the imaging FOV from ~3.6 mm to ~5 mm. The excitation for the handheld FD FLIM system is a 375nm CW diode laser modulated at 1.25MHz and 20MHz. The fluorescence emission is spectrally divided in three emission bands (405/40nm, 440/40nm, and 525/50nm) and detected by three independent APDs. The multispectral signals are further digitized and processed by a FPGA. Phase shift and decreased magnitude are computed at 1.25MHz, 20MHz and its harmonic frequencies (40MHz, 60MHz, 80MHz and 100MHz) via Discrete Fourier Transform (DFT) for lifetime estimation. The current pixel rate is 12.5 KHz which is limited by the SNR. The system is validated by imaging standard fluorescent dyes and human healthy oral mucosa in vivo. This handheld FLIM system offers a cost reduction of at least 50% compared to previous time-domain implementations.
Cancer development in oral epithelial tissue induces subtle changes in tissue autofluorescence that are associated with increased metabolic activity in malignant oral epithelial cells. These autofluorescence biomarkers of oral cancer progression include a decrease in the optical “redox ratio”, defined as the autofluorescence intensity of NADH divided by that of FAD, and specific changes in the fluorescence lifetime of both NADH and FAD. We therefore hypothesized that more specific biomarkers of oral cancer and dysplasia can more accurately be quantified by endogenous fluorescence lifetime imaging (FLIM). In this work, FLIM images of benign, dysplastic and early stage cancerous oral lesions from 52 patients were acquired at three emission channels (390±20nm, 452±22.5nm and >500nm) using a handheld multispectral FLIM endoscope. For each pixel, the fluorescence decays collected at the three emission bands were analyzed using a biexponential decay model, resulting on 16 FLIM-derived parameters per pixel, which generated multiparametric FLIM images of each oral lesion. Statistical analysis was performed on each of the computed FLIM parameters (Wilcoxon test: Normal vs. Benign, Normal vs. Dysplasia/Cancer; Mann-Whitney test: Benign vs. Dysplasia/Cancer). Results from this analysis revealed that FLIM-derived parameters associated with collagen lifetime, NADH lifetime, FAD autofluorescence, and the optical redox ratio were statistically different between dysplastic/cancerous vs. benign oral lesions. This study provides the first demonstration for the clinical imaging of autofluorescence biochemical and metabolic biomarkers of oral epithelial cancer and dysplasia, which could potentially enable early detection of oral cancer.
Despite the fact that the oral cavity is easily accessible, only ~30% of oral cancers are diagnosed at an early stage, which is the main factor attributed to the low 5-year survival rate (63%) of oral cancer patients. Several screening tools for oral cancer have been commercially available; however, none of them have been demonstrated to have sufficient sensitivity and specificity for early detection of oral cancer and dysplasia. We hypothesized that an array of biochemical and metabolic biomarkers for oral cancer and dysplasia can be quantified by endogenous fluorescence lifetime imaging (FLIM), thus enabling levels of sensitivity and specificity adequate for early detection of oral cancer and dysplasia. Our group has recently developed multispectral FLIM endoscopes to image the oral cavity with unprecedented imaging speed (>2fps). We have also performed an in vivo pilot study, in which endogenous multispectral FLIM images were acquired from clinically suspicious oral lesions of 70 patients undergoing tissue biopsy. The results from this pilot study indicated that mild-dysplasia and early stage oral cancer could be detected from benign lesions using a computed aided diagnosis (CAD) system developed based on biochemical and metabolic biomarkers that could be quantified from endogenous multispectral FLIM images. The diagnostic performance of this novel FLIM based clinical tool was estimated using a cross-validation approach, showing levels of sensitivity >90%, specificity >80%, and Area Under the Receiving Operating Curve (RO- AUC) >0.9. Future efforts are focused on developing cost-effective FLIM endoscopes and validating this novel clinical tool in prospective multi-center clinical studies.
Despite the fact that the oral cavity is easily accessible, only ~30% of oral cancers are diagnosed at an early stage, which is the main factor attributed to the low 5-year survival rate (63%) of oral cancer patients. Several screening tools for oral cancer have been commercially available; however, none of them have been demonstrated to have sufficient sensitivity and specificity for early detection of oral cancer and dysplasia. We hypothesized that an array of biochemical and metabolic biomarkers for oral cancer and dysplasia can be quantified by endogenous fluorescence lifetime imaging (FLIM), thus enabling levels of sensitivity and specificity adequate for early detection of oral cancer and dysplasia. Our group has recently developed multispectral FLIM endoscopes to image the oral cavity with unprecedented imaging speed (>2fps). We have also performed an in vivo pilot study, in which endogenous multispectral FLIM images were acquired from clinically suspicious oral lesions of 52 patients undergoing tissue biopsy. The results from this pilot study indicated that mild-dysplasia and early stage oral cancer could be detected from benign lesions using a computed aided diagnosis (CAD) system developed based on biochemical and metabolic biomarkers that could be quantified from endogenous multispectral FLIM images. The diagnostic performance of this novel FLIM clinical tool was estimated using a cross-validation approach, showing levels of sensitivity and specificity >80%, and Area Under the Receiving Operating Curve (RO- AUC) >0.9. Future efforts are focused on developing cost-effective FLIM endoscopes and validating this novel clinical tool in prospective multi-center clinical studies.
Despite of the ease accessibility of the oral cavity, only ~30% of oral cancer patients are diagnosed at early stages. Some of the factors that contribute to this low rate of early detection are: asymptomatic oral cancer lesions, similarity to benign lesions, and sampling error during biopsy procedures. Progression of oral cancer is accompanied by alterations in the intrinsic fluorescence properties of the oral tissue, making fluorescence lifetime imaging (FLIM) suitable for the diagnosis of oral cancer. In this study, in vivo human oral lesions from 70 patients were imaged using a multispectral FLIM endoscopy system. The collected database consisted of 50 benign lesions, and 20 dysplastic and early stage cancerous lesions, as determined by histopathological diagnosis. For each pixel, three fluorescence decays were collected corresponding to three emission bands (390 nm, 450 nm, 500 nm), and analyzed using a biexponential decay model. Selected parameters of this fitting algorithm along with the normalized intensities at each emission band were used as features for a quadratic discriminant analysis (QDA) classifier. The classification performance was estimated using a 10 fold cross-validation approach, resulting on levels of sensitivity and specificity >85%, and an ROC AUC of 0.9 for detecting dysplastic and cancerous oral lesions from benign lesions. These results demonstrate the potential of endogenous FLIM endoscopy for automated early detection of oral cancer.
In this work, we compare standard wide-field fluorescence lifetime imaging microscopy (FLIM) and structured illumination FLIM (SI-FLIM) as methods for the early detection of oral squamous cell carcinoma (OSCC). Our technique, SI-FLIM, provides depth dependent fluorescence lifetime information of the oral epithelium, isolating the endogenous fluorophore of interest, NADH, from interfering fluorescence generated mainly by collagen in the lamina propria. Male golden Syrian hamsters (Cricetus auratus) were used as the animal model for OSCC. They were treated with a carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), for a twelve-week period by applying the DMBA suspended in mineral oil to their cheek pouches 3 times per week. The progression of OSCC was monitored over a 12-week period with imaging beginning at the 6th week. The cheek pouch with lesions was imaged in a 3x4 grid (twelve total images), with each section of the grid being correlated with histopathological analysis. The NADH fluorescence channel, as a diagnostic indicator, was compared for both SI-FLIM and widefield FLIM. ROC analysis, in the task of distinguishing between mild dysplasia and normal tissue, showed that SI-FLIM (AUC=0.83, se=0.07) may be a better indicator for early cases of mild dysplasia when compared to widefield FLIM (AUC=0.63, se=0.07) with statistical significance.
Endogenous fluorescence lifetime imaging (FLIM) provides direct access to the concomitant functional and biochemical changes accompanying tissue transition from benign to precancerous and cancerous. Since FLIM can noninvasively measure different and complementary biomarkers of precancer and cancer, we hypothesize that it will aid in clinically detecting early oral epithelial cancer. Our group has recently demonstrated the detection of benign from premalignant and malignant lesions based on endogenous multispectral FLIM in the hamster cheek-pouch model. Encouraged by these positive preliminary results, we have developed a handheld endoscope capable of acquiring multispectral FLIM images in real time from the oral mucosa. This novel FLIM endoscope is being used for imaging clinically suspicious pre-malignant and malignant lesions from patients before undergoing tissue biopsy for histopathological diagnosis of oral epithelial cancer. Our preliminary results thus far are already suggesting the potential of endogenous FLIM for distinguishing a variety of benign lesions from advanced dysplasia and squamous cell carcinoma (SCC). To the best of out knowledge, this is the first in vivo human study aiming to demonstrate the ability to predict the true malignancy of clinically suspicious lesions using endogenous FLIM. If successful, the resulting clinical tool will allow noninvasive real-time detection of epithelial precancerous and cancerous lesions in the oral mucosa and could potentially be used to assist at every step involved on the clinical management of oral cancer patients, from early screening and diagnosis, to treatment and monitoring of recurrence.
We present the use of a commercially available electrically tunable lens to achieve axial scanning in a reflectance confocal microscope. Over a 255 μm axial scan range, the lateral and axial resolutions varied from 1-2 μm and 4-14 μm, respectively, dependent on the variable focal length of the tunable lens. Confocal imaging was performed on normal human biopsies from the oral cavity ex vivo. Sub-cellular morphologic features were seen throughout the depth of the epithelium while axially scanning using the focus tunable lens.
Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm2 tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1 μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.
A highly sensitive, shot-noise-limited Raman signal acquisition is achieved using frequency-time transformation in a single-mode fiber and time-correlated photon counting system. To spectrally disperse Raman signal excited by a picosecond laser pulse, the light is directed into a sufficiently long single-mode fiber. The output end of the fiber is coupled into a time-gated photon multiplier tube (PMT). Due to a frequency-time conversion provided by the fiber core, photons of different frequencies experience different transient times. In this way, by measuring the photons’ arrival time, Raman peaks can be recorded and separated. Moreover, in some cases the fluorescence background can be eliminated from Raman signals due to its much longer life-time. Consequently, a fluorescent background free Raman spectrum can be attained using the time-correlated photon-counting Raman spectroscopy. In this report, by using a 400m SM600 single-mode fiber and a Hamamatsu R3809U-50 PMT, we demonstrate the Raman spectrum of dimethyl sulfoxide excited by a short-pulsed laser.
Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue
with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation
followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and
objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we
present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance
and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12
mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that
our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can
potentially serve as a guide in early oral cancer detection..
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