SignificanceBreast conservation therapy is the preferred technique for treating primary breast cancers. However, breast tumor margins are hard to determine as tumor borders are often ill-defined. As such, there exists a need for a clinically compatible tumor margin detection system.AimA combined time-resolved fluorescence and diffuse reflectance (TRF-DR) system has been developed to determine the optical properties of breast tissue. This study aims to improve tissue classification to aid in surgical decision making.ApproachNormal and tumor breast tissue were collected from 80 patients with invasive ductal carcinoma and measured in the optical system. Optical parameters were extracted, and the tissue underwent histopathological examination. In total, 761 adipose, 77 fibroglandular, and 347 tumor spectra were analyzed. Principal component analysis and decision tree modeling were performed using only TRF optical parameters, only DR optical parameters, and using the combined datasets.ResultsThe classification modeling using TRF data alone resulted in a tumor margin detection sensitivity of 72.3% and specificity of 88.3%. Prediction modeling using DR data alone resulted in greater sensitivity and specificity of 80.4% and 94.0%, respectively. Combining both datasets resulted in the improved sensitivity and specificity of 85.6% and 95.3%, respectively. While both sensitivity and specificity improved with the combined modeling, further study of fibroglandular tissue could result in improved classification.ConclusionThe combined TRF-DR system showed greater tissue classification capability than either technique alone. Further work studying more fibroglandular tissue and tissue of mixed composition would develop this system for intraoperative use for tumor margin detection.
In endoscopy, the position and orientation of the endoscope’s distal tip are important for locating lesions. Currently, position is estimated using scale lines on the endoscope’s surface and orientation is estimated based on the position of the proximal handle. Both these estimates are inaccurate and cannot be continuously recorded. Real-time endoscope motion tracking should improve tumor localization during colonoscopy. We developed an endoscope tracker that uses a pair of trackballs and two video cameras. It can track real-time changes in insertion length and rotation during an endoscopic procedure. A prototype device has been built and its performance has been evaluated.
Toward objective assessment of skin erythema, diverse approaches were in a contest. Yet, spectral measurement techniques are discriminated by identifying the distinct states of skin health with a unique signature. In this work, we selected two spectral techniques for the assessment of erythema induced by radiation therapy of skin cancer. The selected techniques are diffuse reflectance spectroscopy (DRS) measurements and hyperspectral imaging (HSI). The purpose of this work is to evaluate the performance of DRS and HSI compared with the visual assessment (VA) technique. VA is the gold standard for erythema assessment. For evaluation purposes, erythema indices were computed for both spectral techniques. Next, Pearson correlation was computed relative to VA scores. The results showed that HSI had a higher correlation with VA rather than DRS technique. In sum, the DRS technique suffers from the limited region of measurements and being in contact with the skin which is not the case with HSI.
Combined with confocal imaging, Fluorescence lifetime imaging microscopy (FLIM) can achieve 3-dimensional optical sectional capability with sub-nanosecond lifetime information. As confocal FLIM acquires multi-dimensional data 4D (3D space + time), it is inherently slow. Recent developments in lock-in pixel imagers with time gated pixels show such detectors are capable of collecting as many as 8-time gates in a single pixel cycle. We present a multiplexed confocal FLIM microscope, equipped with a 4-taps time-gated lock-in pixel imager. The multiplexing setup allows the use of the sparse array with sub-nanosecond time-gating to achieve high throughput FLIM acquisition.
Quantitative measurement of protein-protein interaction is important for many biological processes, including cell growth, intercellular communication, gene expression and apoptosis. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET can be measured by changes in fluorescence lifetime of the fluorophors by Fluorescence lifetime imaging microscopy (FLIM) in live cells. Combined with confocal imaging, FLIM can achieve 3-dimensional optical sectional capability and resolve sub-cellular structures. The change in lifetime is inversely proportional to the ratio of bound to non-bound proteins. Time-resolved conventional confocal scanning microscopy is inherently slow and not suitable for rapid imaging applications. We present a 32×32 multiplexing confocal microscope, equipped with a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. The multiplexing setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. We used this multiplexing confocal FLIM system to measure Bcl-2 family proteins interactions in live cells and are able to capture a 240×240 μm FOV multi-channel confocal FLIM images in less than 1.5s. Protein binding affinities are estimated by measuring the changes in FRET as function of acceptor to donor ratio.
Trichomoniasis is one of the most common non-viral sexually transmitted infection caused by a parasite called Trichomonas vaginalis. Currently, there is no timely, cost-efficient diagnostic test exist for Trichomoniasis. We present a lensless optofluidic imaging technique for label-free point-of-care detection of motile parasites in bodily fluids. With this setup we are able to achieve a FOV of ~2 mm2 at a frame rate of 30 fps and can see ~200 μl of sample in one image. Trichomonas vaginalis is identifiable through simple morphological features. Contrary to common practice of minimizing the distance between the sample and the sensor to maximize the resolution, we demonstrated that a greater distance is more advantageous by using the parasites natural ability to focus light as a contrast mechanism. This technique uses a low-cost high frame rate CMOS detector, which enables high throughput operation with requires minimal sample preparation, making it a promising solution to the rapid diagnosis of Trichomoniasis.
KEYWORDS: Data acquisition, Sensors, Humidity, Statistical analysis, Clouds, Temperature metrology, Temperature sensors, Data communications, Environmental sensing, Sensing systems
Experimental data acquisition and statistical data analysis are core components in photonics undergraduate curriculum. Although it focuses on experimental data, the content is usually delivered by a lecture-based format. This is partially because the contents are delivered at the beginning of the program when experimental data acquisition techniques have not yet been introduced. In a second-year data acquisition and applied statistics course, we have designed an experiential learning module that covers the fundamental content of data acquisition and statistical analysis. This module uses a single physical experimental setup that is continuously measuring environmental parameters (temperature, humidity, light, imaging, etc.) using a set of multiple modality sensors in an Internet-of-things (IoT) big data platform (Pi Vision, OSI Soft). Different types of sensors measuring the same parameters are also used for cross-validation purposes. The data is streamed to a cloud computing platform, allowing each student to acquire their own subset of data, and then perform processing and analysis. The capability of remote access a physical sensing experiment provide the students hands-on learning opportunities on a managed complex data acquisition system. The platform provides a set of powerful visualization tools to allow a multi-dimension view of complex data streams (e.g. time-lapse of statistical distribution). Such IoT data acquisition platform allows key concepts to be demonstrated, applied, and tested including error propagation, distribution and test of distribution, correlation and cross-validation, data rejection, and signal processing. This experiential learning module has been demonstrated to be more effective in achieving related learning objectives through quantitative graduate attribute measurements as well as qualitative feedback.
Surveillance and assessment of radiation-induced erythema is an important aspect of managing skin toxicity in radiation therapy treated patients. Upon receiving the early fractions of radiation, an inflammatory response and vascular dilation takes place due to damage of basal cells in the skin’s epidermal layer. This process of skin reddening known as erythema. The gold standard used for assessing and grading erythema is visual assessment (VA) by an experienced clinician/ radiotherapist using toxicity scoring tools. This method is limited by the assessor’s experience, vision acuity, and the subjectivity of qualitative scores. An alternative optical technique to VA, is diffuse reflectance spectroscopy (DRS). A comparison between both techniques performance in detecting radiation therapy-induced erythema is demonstrated in this pilot study. The results evidenced that DRS is capable of detecting skin erythema before an expert eye could do so.
This work describes a procedure to characterize a developed acousto-optic tunable filter (AOTF) based hyperspectral imaging (HSI) system, operating in the visible-near infrared (VNIR) spectrum. The developed AOTF-HSI system consists of an AOTF (11 × 12 mm aperture), a set of optics, and a computer. The AOTFHSI setup includes a complementary metal oxide semiconductor (CMOS) camera (2048 x 2048 pixels), a zoom lens (55–250 mm f/4–5.6), and an AOTF radio frequency synthesizer. Two multifaceted reflector tungstenhalogen lamps (150 W) were used to provide double-sided illumination to the region of interest. Image acquisition was accomplished by a home-made C# code. The code enables imaging of samples in the VNIR (450 – 850 nm) range in both hyper/multi-spectral modes. The developed spectral imager presents a valuable opportunity for noninvasive evaluation of medical samples.
Ultrashort lasers are promising tools for surgical and dental applications where precise cutting of hard tissue is required with minimal collateral tissues damage. We report the use of an amplified femtosecond laser (800 nm, 210 fs, 1 kHz, and up to 200 µJ) to generate high aspect ratio structures on bovine bone samples. The ablation fluence threshold of bovine bone was determined and the incubation effect was observed. The incubation coefficient and the single-pulse ablation threshold were calculated to be 0.90±0.02 and 1.43±0.09 J/cm2, respectively. The influence of experimental conditions on laser ablation were also investigated by performing craters with dimensions of 1000 x 220 µm under dry, compressed air, and flowing water treatments during ablation. Our results show that compressed air treatment produced the lower roughness of crater walls as well as highest depth, compared with those of dry and water conditions. The craters produced in dry and compressed air treatment have a “V” shape, while a “U” shape was observed when using flowing water treatment.
In this work, we report a performance comparison of an acousto-optic tunable filter (AOTF), and a liquid crystal tunable filter (LCTF) based on a novel dual-arm hyperspectral imaging (HSI) configuration. The main purpose of this work is to highlight the leverage points of each tunable filter, in order to facilitate filter choice in HSI design. Three main parameters are experimentally examined: spectral resolution, out-of-band suppression, and image quality in the sense of spatial resolution. The experimental results, using wideband illumination, laser lines, and a spatial test target (USAF-1951) emphasized the superiority of AOTF in spectral resolution, out-of –band suppression and random switching speed between wavelengths. The same experiments demonstrated LCTF to have better performance in terms of the spatial image resolution, both horizontal and vertical, and high definition quality. In conclusion, the efficient design of an HSI system is application-dependent. For medical applications, for instance, if the tissue of interest has undefined optical properties, or contains close spectral features, AOTF might be the better option. Otherwise, LCTF is more convenient and simpler to use, especially if the tissue chromophore’s spatial mapping is needed.
Visual assessment is the most common clinical investigation of skin reactions in radiotherapy. Due to the subjective nature of this method, additional noninvasive techniques are needed for more accurate evaluation. Our goal is to evaluate the effectiveness of hyperspectral image analysis for that purpose. In this pilot study, we focused on detection and grading of skin Erythema. This paper reports our proposed processing pipeline and experimental findings. Experiments have been performed to demonstrate the efficacy of the proposed approach for (1) reproducing clinical assessments, and (2) outperforming RGB imaging data.
Glioma itself accounts for 80% of all malignant primary brain tumors, and glioblastoma multiforme (GBM) accounts for 55% of such tumors. Diffuse reflectance and fluorescence spectroscopy have the potential to discriminate healthy tissues from abnormal tissues and therefore are promising noninvasive methods for improving the accuracy of brain tissue resection. Optical properties were retrieved using an experimentally evaluated inverse solution. On average, the scattering coefficient is 2.4 times higher in GBM than in low grade glioma (LGG), and the absorption coefficient is 48% higher. In addition, the ratio of fluorescence to diffuse reflectance at the emission peak of 460 nm is 2.6 times higher for LGG while reflectance at 650 nm is 2.7 times higher for GBM. The results reported also show that the combination of diffuse reflectance and fluorescence spectroscopy could achieve sensitivity of 100% and specificity of 90% in discriminating GBM from LGG during ex vivo measurements of 22 sites from seven glioma specimens. Therefore, the current technique might be a promising tool for aiding neurosurgeons in determining the extent of surgical resection of glioma and, thus, improving intraoperative tumor identification for guiding surgical intervention.
The ability to recover the intrinsic fluorescence of biological fluorophores is crucial to accurately identify the fluorophores and quantify their concentrations in the media. Although some studies have successfully retrieved the fluorescence spectral shape of known fluorophores, the techniques usually came with heavy computation costs and did not apply for strongly absorptive media, and the intrinsic fluorescence intensity and fluorophore concentration were not recovered. In this communication, an experimental approach was presented to recover intrinsic fluorescence and concentration of fluorescein in the presence of hemoglobin (Hb). The results indicated that the method was efficient in recovering the intrinsic fluorescence peak and fluorophore concentration with an error of 3% and 10%, respectively. The results also suggested that chromophores with irregular absorption spectra (e.g., Hb) have more profound effects on fluorescence spectral shape than chromophores with monotonic absorption and scattering spectra (e.g., black India ink and polystyrene microspheres).
Early detection and treatment of high-grade dysplasia (HGD) in Barrett’s esophagus may reduce the risk of developing esophageal adenocarcinoma. Confocal endomicroscopy (CLE) has shown advantages over routine white-light endoscopic surveillance with biopsy for histological examination; however, CLE is compromised by insufficient contrast and by intra- and interobserver variation. An FDA-approved PDT photosensitizer was used here to reveal morphological and textural features similar to those found in histological analysis. Support vector machines were trained using the aforementioned features to obtain an automatic and robust detection of HGD. Our results showed 95% sensitivity and 87% specificity using the optimal feature combination and demonstrated the potential for extension to a three-dimensional cell model.
High-grade dysplasia (HGD) in Barrett’s esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the “seek-and-treat” potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time “seek-and-treat” strategies in three-dimensional models for improving early detection and treatment of BE.
Optical biopsy techniques offer a minimally invasive, real-time alternative to traditional biopsy and pathology during tumor resection surgery. Diffuse reflectance spectroscopy (DRS) is a commonly used technique in optical biopsy. Optical property recovery from spatially resolved DRS data allows quantification of the scattering and absorption properties of tissue. Monte Carlo simulation methods were used to evaluate a unique fiber-optic probe design for a DRS instrument to be used specifically for optical biopsy of the brain. The probe diameter was kept to a minimum to allow usage in small surgical cavities at least 1 cm in diameter. Simulations showed that the close proximity of fibers to the edge of the probe resulted in boundary effects due to reflection of photons from the surrounding air–tissue interface. A new algorithm for rapid optical property recovery was developed that accounts for this reflection and therefore overcomes these effects. The parameters of the algorithm were adjusted for use over the wide range of optical properties encountered in brain tissue, and its precision was evaluated by subjecting it to random noise. This algorithm can be adapted to work with any probe geometry to allow optical property recovery in small surgical cavities.
A hyperspectral fluorescence lifetime imaging (FLIM) instrument is developed to study endogenous fluorophores in biological tissue as an optical biopsy tool. This instrument is able to spectrally, temporally, and spatially resolve fluorescence signal, thus providing multidimensional information to assist clinical tissue diagnosis. An acousto-optic tunable filter (AOTF) is used to realize rapid wavelength switch, and a photomultiplier tube and a high-speed digitizer are used to collect the time-resolved fluorescence decay at each wavelength in real time. The performance of this instrument has been characterized and validated on fluorescence tissue phantoms and fresh porcine skin specimens. This dual-arm AOTF design achieves high spectral throughput while allowing microsecond nonsequential, random wavelength switching, which is highly desirable for time-critical applications. In the results reported here, a motorized scanning stage is used to realize spatial scanning for two-dimensional images, while a rapid beam steering technique is feasible and being developed in an ongoing project.
When using ultrafast laser ablation in some orthopedic applications where precise cutting/drilling is required with minimal damage to collateral tissue, it is challenging to produce large-sized and deep holes using a tightly focused laser beam. The feasibility of producing deep, millimeter-size structures under different ablation strategies is investigated. X-ray computed microtomography was employed to analyze the morphology of these structures. Our results demonstrated the feasibility of producing holes with sizes required in clinical applications using concentric and helical ablation protocols.
Ultrashort pulsed lasers in bone ablation show promise for many orthopedic applications. To minimize collateral tissue damage and control the ablation process, the ablation threshold fluence must be well characterized. Using an amplified femtosecond laser (170 fs, 800 nm, 1 kHz), the ablation threshold on unaltered porcine cortical bone was measured using the D2 method at multiple incident pulse numbers ranging from 25 to 1000 pulses per spot. The lowered threshold at greater pulse numbers indicated an incubation effect. Using a power law model, the incubation coefficient of unaltered porcine cortical bone was found to be 0.89 ± 0.03. Through extrapolation, the single-pulse ablation threshold was found to be 3.29 ± 0.14 J/cm2.
KEYWORDS: Endoscopes, Mirrors, Modulation transfer functions, Prototyping, Lens design, Combined lens-mirror systems, Objectives, Monte Carlo methods, Endoscopy, Imaging systems
Autofluorescence endoscopy is a promising functional imaging technique to improve screening of pre-cancerous or early cancer lesions in the gastrointestinal (GI) tract. Tissue autofluorescence signal is weak compared to white light reflectance imaging. Conventional forward-viewing endoscopes are inefficient in the collection of light from objects of interest along on the GI luminal wall. A key component of a complete autofluorescence endoscope is the light collection module. In this paper, we report the design, optimization, prototype development, and testing of an endoscope objective that is capable of acquiring simultaneous forward and radial views. The radial-view optical design was optimized for a balance between image quality and light collection. Modulation transfer function (MTF), entrance pupil radius, manufacturability, and field-of-view were parameters used in the lens optimization. In comparison with the typical forward-viewing endoscopes, our nonsequential ray trace simulations suggest the proposed radial-view design is more practical in the light collection. To validate the proposed simulation methods, a 3:1 scaled-up prototype was fabricated. Contrast measurements were taken with the prototype, and then compared with the simulated MTF.
The goal of this study is to determine the potential of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) as an adjunctive tool for delineation of brain tumor from surrounding normal tissue in order to assist the neurosurgeon in near-complete tumor excision. A time-domain TR-LIFS prototype apparatus (gated photomultiplier detection, fast digitizer) was used for recording tissue autofluorescence in normal cortex (NC), normal white matter (NWM), and various grades of gliomas intraoperatively. Tissue fluorescence was induced with a pulsed nitrogen laser (337 nm, 700 ps), and the intensity decay profiles were recorded in the 360- to 550-nm spectral range (10-nm interval). Histopathological analysis (hematoxylin & eosin) of the biopsy samples taken from the site of TR-LIFS measurements was used for validation of spectroscopic results. Preliminary results on 17 patients demonstrate that normal cortex (N=16) and normal white matter (N=3) show two peaks of fluorescence emission at 390 nm (lifetime=1.8±0.3 ns) and 460 nm (lifetime=0.8±0.1 ns). The 390-nm emission peak is absent in low-grade glioma (N=5; lifetime=1.1 ns) and reduced in high-grade glioma (N=9; lifetime=1.7±0.4 ns). The emission characteristics at 460 nm in all tissues correlated with the nicotinamide adenine dinucleotide fluorescence (peak: 440 to 460 nm; lifetime: 0.8 to 1.0 ns). These findings demonstrate the potential of using TR-LIFS as a tool for enhanced delineation of brain tumors during surgery. In addition, this study evaluates similarities and differences between TR-LIFS signatures of brain tumors obtained in vivo and those previously reported in ex vivo brain tumor specimens.
KEYWORDS: Time resolved spectroscopy, Luminescence, Statistical analysis, Principal component analysis, Tissues, Surface plasmons, In vivo imaging, Deconvolution, Biological research, Spectroscopy
We report the application of the Laguerre deconvolution technique (LDT) to the analysis of in-vivo time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data and the diagnosis of atherosclerotic plaques. TR-LIFS measurements were obtained in vivo from normal and atherosclerotic aortas (eight rabbits, 73 areas), and subsequently analyzed using LDT. Spectral and time-resolved features were used to develop four classification algorithms: linear discriminant analysis (LDA), stepwise LDA (SLDA), principal component analysis (PCA), and artificial neural network (ANN). Accurate deconvolution of TR-LIFS in-vivo measurements from normal and atherosclerotic arteries was provided by LDT. The derived Laguerre expansion coefficients reflected changes in the arterial biochemical composition, and provided a means to discriminate lesions rich in macrophages with high sensitivity (>85%) and specificity (>95%). Classification algorithms (SLDA and PCA) using a selected number of features with maximum discriminating power provided the best performance. This study demonstrates the potential of the LDT for in-vivo tissue diagnosis, and specifically for the detection of macrophages infiltration in atherosclerotic lesions, a key marker of plaque vulnerability.
The overall objective this work is the development of a miniaturized fluorescence spectroscopy analyzer realized via microfabrication technology. Previously, we reported a MEMS micro grating actuated by a piezoelectric cantilever. For such device to be used in a spectroscopic system, optical characterization of the grating's efficiency and the system's stray light are required. We report here the characterization of the grating cantilever with a MEMS micro lens with the intention of fitting into a packaged micro spectroscopic system. This packaging is accomplished by multi-wafer (silicon) bonding of strategically aligned crystalline planes in order to form the basic geometry of a miniaturized spectroscopy setup. One of these crystalline planes, <111> of silicon, is used as a mirror for folding and compacting the optics at the specific angle of 54.74° (with wafer plane normal). The packaging, microlens, and grating cantilever are position in the designed geometry to accept a self-aligned fiber input from a flash lamp source. The microlens component is presented with beam profilometry of its focusing at a focal length of 7.7 mm. The diffraction is interrogated by a monochromator for quantifying the above said characteristics. The relative efficiency of the grating was 40-70% in the 400-600 nm range. Together these characterized components define the geometry and performance of our micro fluorescence spectroscopy system.
KEYWORDS: Luminescence, Deconvolution, Tissues, Spectroscopy, Laser induced fluorescence, Inflammation, Fluorescence spectroscopy, Time resolved spectroscopy, Data analysis, In vivo imaging
This study introduces new methods of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data analysis for tissue characterization. These analytical methods were applied for the detection of atherosclerotic vulnerable plaques. Upon pulsed nitrogen laser (337 nm, 1 ns) excitation, TR-LIFS measurements were obtained from carotid atherosclerotic plaque specimens (57 endarteroctomy patients) at 492 distinct areas. The emission was both spectrally- (360-600 nm range at 5 nm interval) and temporally- (0.3 ns resolution) resolved using a prototype clinically compatible fiber-optic catheter TR-LIFS apparatus. The TR-LIFS measurements were subsequently analyzed using a standard multiexponential deconvolution and a recently introduced Laguerre deconvolution technique. Based on their histopathology, the lesions were classified as early (thin intima), fibrotic (collagen-rich intima), and high-risk (thin cap over necrotic core and/or inflamed intima). Stepwise linear discriminant analysis (SLDA) was applied for lesion classification. Normalized spectral intensity values and Laguerre expansion coefficients (LEC) at discrete emission wavelengths (390, 450, 500 and 550 nm) were used as features for classification. The Laguerre based SLDA classifier provided discrimination of high-risk lesions with high sensitivity (SE>81%) and specificity (SP>95%). Based on these findings, we believe that TR-LIFS information derived from the Laguerre expansion coefficients can provide a valuable additional dimension for the diagnosis of high-risk vulnerable atherosclerotic plaques.
In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.
It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.
KEYWORDS: Luminescence, Time resolved spectroscopy, In vivo imaging, Fiber optics, Foam, Fluorescence spectroscopy, Data acquisition, Tissues, Signal to noise ratio, Spectroscopes
Time-resolved laser-induced fluorescence spectroscopy (tr-LIFS) has been studied as a potential tool for in vivo diagnosis of atherosclerotic lesions. This study is to evaluate the potential of a compact fiber-optics based tr-LIFS instrument developed in our laboratory for in vivo analysis of atherosclerotic plaque composition. Time-resolved fluorescence spectroscopy studies were performed in vivo on fifteen New Zealand White rabbits (atherosclerotic: N=8, control: N=7). Time-resolved fluorescence spectra were acquired (range: 360-600 nm, increment: 5 nm, total acquisition time: 65 s) from normal aorta wall and lesions in the abdominal aorta. Data were analyzed in terms of fluorescence emission spectra and wavelength specific lifetimes. Following trichrome staining, tissue specimens were analyzed histopathologically in terms of intima/media thickness and biochemical composition (collagen, elastin, foam cells, and etc). Based on intimal thickness, the lesions were divided into thin and thick lesions. Each group was further separated into two categories: collagen rich lesions and foam cell rich lesions based on their biochemical composition. The obtained spectral and time domain fluorescence signatures were subsequently correlated to the histopathological findings. The results have shown that time-domain fluorescence spectral features can be used in vivo to separate atherosclerotic lesions from normal aorta wall as well discrimination within certain types of lesions.
To estimate the intrinsic fluorescence intensity decay of a compound, the excitation light pulse must be deconvolved from the measured fluorescence pulse trace. The most commonly used deconvolution method is the multiexponential least-square iterative reconvolution (LSIR) technique. A variant of LSIR in which the intrinsic fluorescence intensity decay is expressed as an expansion on the discrete time Laguerre basis, was recently introduced. In this study, the performance of the Laguerre deconvolution technique was successfully tested with simulated and fluorescence standard data. It was also demonstrated that the Laguerre deconvolution presents a number of advantages over the classical multiexponential LSIR, including less expensive computational resolution, and the property to generate a unique set of expansion coefficients highly correlated with the intrinsic lifetimes. A novel method for concentration estimation based on the analysis of the Laguerre expansion coefficients was also proposed and successfully applied to different fluorescence standard mixtures, performing even better (error<2%) than more traditional methods of spectral analysis, such as PCR (error<7%) and PLS (error<10%). These findings suggest that the use of Laguerre expansion coefficients represents an alternative nonparametric approach to characterize and discriminate biological systems, in terms of their spectral and lifetime characteristics.
For complex biological systems, conventional analysis of fluorescence intensity decay in terms of discrete exponential components cannot readily provide a true representation of the underlying fluorescence dynamics. We investigate an alternative nonparametric method for the analysis of time-resolved fluorescence data from biochemical and biological systems based on the expansion of fluorescence decay in a discrete Laguerre basis. We report that a unique Laguerre expansion can be found for fluorescence intensity decays of arbitrary form with convergence to a correct solution significantly faster than conventional multiexponential approximation methods. The Laguerre expansion coefficients are shown to be highly correlated with intrinsic fluorescence lifetimes and allow direct characterization of the fluorescence dynamics. A novel method for prediction of concentrations in mixtures of biochemical components using these coefficients is developed and successfully tested (prediction error <2%) using data from different mixtures of fluorescence lifetime standards. These findings suggest that the use of Laguerre expansion coefficients is a fast approach for the characterization and discrimination of complex biological systems such as tissues and cells, and that the method has potential for applications of fluorescence lifetime techniques to tissue diagnostics and imaging microscopy of living cells.
We report on the design, development, and performance of a versatile and mobile fiber-optic based time-resolved fluorescence apparatus for in-vivo spectroscopy of biological tissues. The apparatus is of modular design which allows for facile interfacing with different pulsed light sources and fiber-optic probes. Main features of the apparatus include: 1) fast acquisition of fluorescence decays with high temporal resolution (0.3 ns) for a broad range of emission wavelengths (300 to 850 nm) and lifetimes (0.3 ns to ms), 2) near real time analysis of fluorescence decays via a Laguerre deconvolution technique, and 3) compatibility with the clinical environment (endoscopic procedures or other intraoperative uses). The apparatus is currently employed for in-vivo studies of diseased tissues (cardiovascular and oncological applications).
In this communication, we report the design, development, calibration, and characterization of a fluorescence spectroscopy instrument capable of measuring time-resolved fluorescence spectra from 350nm to 800nm with subnanosecond resolution. The compact system can be used in a variety of clinical settings including endoscopic procedures. The instrument can accommodate various types of laser sources and fiber optic probes for different types of applications. The typical acquisition time is about 0.8 s per wavelength or 40 s for a 200 nm time-resolved fluorescence spectrum including online fluorescence lifetime analysis. The system has a variable spectral resolution from 0.1 to 10 nm and temporal resolution of 300 ps. Fluorescence spectra can be acquired with a sensitivity of 10-9M at signal-to-noise ratio of 46. The fluorescence lifetimes of Rhodamin B and 9- cyanoanthracene were determined to be 2.92∓0.12 ns and 12.28∓0.12 ns indicating good agreement with literature values.
The design of fiber-optic probes plays an important role in optical spectroscopic studies, including fluorescence spectroscopy of biological tissues. It can affect the light delivery and propagation into the tissue, the collection efficiency (total number of photons collected vs. total number of photons launched) and the origin of collected light. This in turn affects the signal to noise ratio (SNR) and the extend of tissue interrogation, thus influencing the diagnostic value of such techniques. Three specific fiber-optic probe designs were tested both experimentally and computationally
via Monte Carlo simulations. In particular, the effects of probe architecture (single-fiber vs. two bifurcated multifiber probes), probe-to-target distance (PTD), and source-to-detector separation (SDS) were investigated on the collected diffuse reflectance of a Lambertian target and an agar-based tissue phantom. This study demonstrated that probe architecture, PTD, and SDS are closely intertwined and considerably affect the light collection efficiency, the extend of target illumination, and the origin of the collected reflected light. Our findings can be applied towards optimization of
fiber-optic probe designs for quantitative fluorescence spectroscopy of diseased tissues.
Treatment of pigmented lesions in skin with visible or near- infrared nanosecond (ns) laser pulses often causes significant collateral tissue damage because the current approach uses pulses with energy of 300 mJ or larger. Additionally, this requires large Q-switched laser systems. To overcome these disadvantages, we have investigated a different approach in delivering ns laser pulses for cutaneous lesion treatment. Tattoo removal in an animal model with a focused laser beam from a Q-switched Nd:YAG laser has been investigated in two Yucatan micropigs tattooed with blue, black, green and red pigments. The tattoos were treated with a focused beam of 12-ns pulses at 1064 nm, with different depth under the skin surface, while the micropig was translated to achieve an effect of single pulse per ablation site in the skin. With the pulse energy reduced to a range from 38 to 63 mJ, we found that nearly complete clearance was achieved for blue and black tattoos while clearance of red and green tattoos was incomplete. Analysis of the skin appearance suggested that the pulse energy can be decreased to below 20 mJ which may lead to further reduction of the collateral tissue damage and improve the clearance of red and green tattoos.
The ablation of porcine skin tissue has been investigated using nanosecond (ns) laser pulses at the wavelengths of 1064, 532 and 266 nm. The ablation probability has been measured near the threshold through detection of the secondary radiation from the tissue sample surface at different wavelengths. Experimental results have indicated that the ablation of the skin tissue in the wide range of ablating wavelength is caused by optical breakdown induced by the strong electromagnetic field of the nanosecond pulses. Furthermore, we conclude that the initial seed electrons acquire ionization energy from the incident optical field mainly through a momentum-relaxing drift mechanism.
In this paper, we report a new effect -- the threshold effect of incident light intensity for the photorefractive light-induced scattering in LiNbO3:Fe, Mg crystals, which could be used as a simple, effective technique to suppress the photorefractively light-induced scattering and is useful for us to get the noise-free photorefractive devices.
In this paper, we introduce a new technology for picking-up images and one-way aberration- free image communication through a phase-disturbing medium using photorefractive four- wave mixing and theoretically analyze this photorefractive process using the photorefractive four-wave coupling equations. We also experimentally realize this operation in the LiNbO3:Fe crystal.
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