Escherichia coli bacteria are a source of food related illness. If irrigation water is contaminated by fecal matter runoff, crops may become infected prior to harvesting, processing, or packaging. Existing test methods require 16-48 hours for confirmation of bacterial infection in the irrigation water. Therefore, providing a means for a rapid detection of water borne coliform and E. coli within a typical workday of 8-10 hours would allow a preventative response. We have developed a method to determine bacteria presence by a measure of metabolic activity with a spectral analysis system. Metabolic activity of live bacteria will appear as a drop in solution pH in a relatively short time frame during the growth phase of the cultured bacteria. A blue LED is used to excite fluorescein fluorescence in the bacterial growth media. The fluorescence exhibits pH sensitive spectral properties within a range of pH 4-7. Unmixing of the fluorescence spectral profile yields the pH and confirms a growing bacteria culture. Results can be provided in hours instead of days, depending on the initial concentration of living bacteria.
We have developed a new tool to measure the acid production by plaque oral bacteria. Many species of oral bacteria metabolize sugars in food and produce organic acids that demineralize the dental enamel leading to the formation of cavities. Measuring the acidity level before and after a sugar rinse can indicate the susceptibility of an individual to tooth decay and location of active caries. In a case study on two subjects, a non-contact optics-based pH device was able to track pH before and after a sugar rinse. The fiber optic probe measures acidity level in difficult to access dental locations such as occlusal pits and fissures based on changes in the spectral fluorescence profile of fluorescein (FL) dye.
Fiber coupled 420 nm LED excites 200uM aqueous FL solution in the mouth. The fluorescence spectrum in 450-650 nm range is obtained using an adjacent fiber optic cable coupled to a spectrometer. Chemometric analysis of endmember dianion and anion species using least-square fitting is performed to determine the pH of the FL absorbed into the extracellular region of the oral biofilm. Other unwanted noise, like background light and auto-fluorescence in the range of 450-650 nm is removed before calculating biofilm pH.
Using this device in a darkened room on two subjects, we were able to measure resting pH (before a sugar rinse) and track time dependent change in pH (after a sugar rinse) in the range of pH 4-7 paving the way for first clinical optical pH measurement in the mouth.
Sugar-rich diets and poor dental hygiene promote the formation of a biofilm (plaque) that strongly adheres to the dental enamel surface and fosters the evolution of aciduric bacteria. The acid contributes to demineralization of the exterior tooth enamel, which accelerates after the pH drops below a critical value (∼5.5) for extended time periods resulting in the need for restorative procedures. Preventative techniques to alert the dentist and caries-susceptible patients regarding vulnerability to dental decay require a clinical measure of plaque activity. Therefore, there is a need to evaluate the acid production capability of plaque deposits in the pits and fissures of occlusal and interproximal regions. A ratiometric fluorescence pH-sensing device has been developed using an FDA-approved dye and LED excitation. Fluorescein spectral profiles were collected using a spectrometer and analyzed with a spectral unmixing algorithm for calibration over the pH range of 4.5 to 7. An in vivo pilot study on human subjects was performed using a sucrose rinse to accelerate bacterial metabolism and to measure the time-dependent drop in pH. The optical system is relatively immune to confounding factors such as photobleaching, dye concentration, and variation in excitation intensity associated with earlier dye-based pH measurement techniques.
A pH measurement of oral biofilms is helpful for monitoring the impact of acidogenic bacteria in the caries process. Demineralization of dental enamel is closely related to the time dependent pH of human plaque. Therefore, providing a means to easily measure the local pH of biofilms is a useful clinical diagnostic in the arsenal of caries prevention tools. Optical measurement methods of plaque metabolism can use intrinsic fluorescence or extrinsic fluorescence from added dyes. Autofluorescence spectral features of human oral biofilms at green (500 nm) and red (634 nm) fluorescence wavelengths using 405 nm excitation did not demonstrate a spectral or intensity shift between neutral and acidic conditions. Chlorin e6, an ingredient in chlorophyllin food supplement, exhibited a spectral and intensity shift of fluorescence emission in buffered solutions, but this quantitative pH-dependence was not transferable to a human plaque environment. Finally, a ratiometric quantitative pH measure was achieved by exciting (405 nm laser) a mixture of two dyes, fluorescein and rhodamine B. This two-dye mixture produced two strong fluorescent bands centered at 515 nm (fluorescein) and 580 nm (rhodamine B), where the 515 nm band was pH sensitive and the 580 nm band served as a pH insensitive reference. This dual-dye fluorescence ratio exhibited a linear response over pH 7 to 5 in human oral biofilms during a sugar challenge. We have explored methods to use non-contact, optical measures of local acidity levels in difficult to access dental locations such as occlusal fissures using various pH sensitive fluorescent dye systems.
An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used in a case study to locate plaque and caries. The imaging system incorporated software mitigation of background auto-fluorescence (AF). In conventional fluorescence imaging, varying AF across a tooth surface can mask low-level porphyrin signals. Laser-induced auto-fluorescence signals of dental tissue excited using a 405-nm laser typically produce fluorescence over a wavelength range extending from 440-nm to 750-nm. Anaerobic bacterial metabolism produces various porphyrin species (eg. protoporphyrin IX) that are located in carious enamel, dentin, gingivitis sites, and plaque. In our case study, these porphyrin deposits remained as long as one day after prophylaxis. Imaging the tooth surface using 405-nm excitation and subtracting the natural AF enhances the image contrast of low-level porphyrin deposits, which would otherwise be masked by the high background AF. In a case study, healthy tissues as well as sites of early and advanced caries formations were scanned for visual and quantitative signs of red fluorescence associated with porphyrin species using a background mitigation algorithm. Initial findings show increasing amplitudes of red fluorescence as caries severity increases from early to late stages. Sites of plaque accumulation also displayed red fluorescence similar to that found in carious dental tissue. The use of real-time background mitigation of natural dental AF can enhance the detection of low porphyrin concentrations that are indicators of early stage caries formation.
Fluorescence molecular imaging with exogenous probes improves specificity for the detection of diseased tissues by targeting unambiguous molecular signatures. Additionally, increased diagnostic sensitivity is expected with the application of multiple molecular probes. We developed a real-time multispectral fluorescence-reflectance scanning fiber endoscope (SFE) for wide-field molecular imaging of fluorescent dye-labeled molecular probes at nanomolar detection levels. Concurrent multichannel imaging with the wide-field SFE also allows for real-time mitigation of the background autofluorescence (AF) signal, especially when fluorescein, a U.S. Food and Drug Administration approved dye, is used as the target fluorophore. Quantitative tissue AF was measured for the ex vivo porcine esophagus and murine brain tissues across the visible and near-infrared spectra. AF signals were then transferred to the unit of targeted fluorophore concentration to evaluate the SFE detection sensitivity for sodium fluorescein and cyanine. Next, we demonstrated a real-time AF mitigation algorithm on a tissue phantom, which featured molecular probe targeted cells of high-grade dysplasia on a substrate containing AF species. The target-to-background ratio was enhanced by more than one order of magnitude when applying the real-time AF mitigation algorithm. Furthermore, a quantitative estimate of the fluorescein photodegradation (photobleaching) rate was evaluated and shown to be insignificant under the illumination conditions of SFE. In summary, the multichannel laser-based flexible SFE has demonstrated the capability to provide sufficient detection sensitivity, image contrast, and quantitative target intensity information for detecting small precancerous lesions in vivo.
Fluorescence-labeled molecular probes can be used during endoscopy for early cancer detection. As many tumors express multiple cell surface markers and these molecular signatures are heterogeneous across patients, simultaneous imaging of numerous different molecular targets is important for increasing the sensitivity of early cancer diagnosis and personalized treatment. For this purpose, a wide-field, multi-spectral fluorescence-reflectance scanning fiber endoscope (SFE) has been developed. Using a set of calibrated fluorescent test targets at in vivo dye concentration, algorithms and methodologies were developed and demonstrated. Preliminary results showed the promise of fluorescence molecular imaging in clinical applications using the multi-spectral SFE.
We developed a multispectral fluorescence-reflectance scanning fiber endoscope (SFE) for wide-field molecular imaging
of fluorescence-labeled molecular probes. Concurrent multi-channels imaging with the wide-field SFE also allows for
real-time mitigation of background autofluorescence (AF) signal, especially when the FDA approved fluorescein is used
as the target fluorophore. In the current study, we demonstrated a real-time AF mitigation algorithm on a tissue phantom
which featured molecular probe targeted cells of high grade dysplasia on a substrate containing AF species. The targetto-
background ratio was enhanced by over an order of magnitude when applying the real-time AF mitigation algorithm.
By minimizing the background signal, multispectral fluorescence imaging can provide sufficient image contrast and
quantitative target information for detecting small pre-cancerous lesions in vivo.
Previously we developed an ultrathin, flexible, multimodal scanning fiber endoscope (SFE) for concurrent white light and fluorescence imaging. Autofluorescence (AF) arising from endogenous fluorophores (primarily collagen in the esophagus) act as major confounders in fluorescence-aided detection. To address the issue of AF, a real-time mitigation algorithm was developed and has been show to successfully remove AF during SFE imaging. To test our algorithm, we previously developed flexible, color-matched, synthetic phantoms featuring a homogenous distribution of collagen. In order to more rigorously test the AF mitigation algorithm, a phantom that better mimicked the in-vivo distribution of collagen in tissue was developed.
The current rise in childhood caries worldwide has increased the demand for portable technologies that can quickly and
accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed
with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype for
detecting and diagnosing occlusal caries demineralization in vivo has been developed and pilot tested. The device uses a
405-nm laser as a scanned illumination source to obtain high resolution and high surface contrast reflectance images,
which allows the user to quickly image and screen for any signs of demineralized enamel. When a suspicious region is
located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser
excitations. These spectra are used to compute an auto-fluorescence (AF) ratio of the suspicious region and the percent
difference of AF ratios from a healthy region of the same tooth. The device was tested on 7 children’s teeth in vivo with
clinically diagnosed carious lesions. Lesion depth was then visually estimated from the video image using the 405-nm
scanned light source, and within a month the maximum drill depth was assessed by a clinician. The researcher and
clinicians were masked from previous measurements in a blinded study protocol. Preliminary results show that the
ratiometric percent difference measurement of the AF spectrum of the tooth correlates with the severity of the
demineralization as assessed by the clinician after drilling.
There is currently a need for a safe and effective way to detect and diagnose early stages of childhood caries. A multimodal optical clinical prototype for diagnosing caries demineralization in vivo has been developed. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and high-contrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. When a suspicious region of demineralization is located, the device also performs dual laser fluorescence spectroscopy using 405- and 532-nm laser excitation. An autofluorescence ratio of the two excitation lasers is computed and used to quantitatively diagnose enamel health. The device was tested on five patients in vivo as well as on 28 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images that highlighted the lesions identified by the clinicians. The autofluorescence spectroscopic ratios obtained from the extracted teeth successfully quantitatively discriminated between sound and demineralized enamel.
KEYWORDS: Luminescence, Phase modulation, Chromium, Signal detection, Multispectral imaging, Endoscopy, Reflectivity, In vivo imaging, Detection and tracking algorithms, Esophagus
The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated.
There is currently a need for a safe and effective way to detect and diagnose early childhood caries. We have developed a multimodal optical clinical prototype for testing in vivo. The device can be used to quickly image and screen for any signs of demineralized enamel by obtaining high-resolution and highcontrast surface images using a 405-nm laser as the illumination source, as well as obtaining autofluorescence and bacterial fluorescence images. Then, when a suspicious region is located, the device can perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitation which is used to compute an autofluorescence ratio. This ratio can be used to quantitatively diagnose enamel health. The device is tested on four in vivo test subjects as well as 17 extracted teeth with clinically diagnosed carious lesions. The device was able to provide detailed images which served to screen for suspected early caries. The autofluorescence ratios obtained from the extracted teeth were able to discriminate between healthy and unhealthy enamel. Therefore, the clinical prototype demonstrates feasibility in screening for and in quantitatively diagnosing healthy from demineralized enamel.
In the present study, artificial type I and type II erosions were created on dental specimen using acetic acid and EDTA respectively. Specimens were prepared by etching extracted teeth samples in acid to varying degrees, after which the absolute fluorescence intensity ratio of the etched enamel relative to sound enamel was recorded for each specimen using 405 and 532 nm laser excitation. Results showed differences in the fluorescence ratio of etched to sound enamel for type I and II erosions. These findings suggest a non-uniform distribution of fluorescent species in the interprismatic region as compared to the prismatic region.
We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic
molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body
movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were
simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent
biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated
using a 1.2mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.
We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2 mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.
An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used to image dental occlusal surfaces as well as shallow artificially induced enamel erosions from human extracted teeth (n = 40). Enhanced image resolution of occlusal surfaces was obtained using a short-wavelength 405-nm illumination laser. In addition, artificial erosions of varying depths were also imaged with 405-, 404-, 532-, and 635-nm illumination lasers. Laser-induced autofluorescence images of the teeth using 405-nm illumination were also obtained. Contrast between sound and eroded enamel was quantitatively computed for each imaging modality. For shallow erosions, the image contrast with respect to sound enamel was greatest for the 405-nm reflected image. It was also determined that the increased contrast was in large part due to volume scattering with a smaller component from surface scattering. Furthermore, images obtained with a shallow penetration depth illumination laser (405 nm) provided the greatest detail of surface enamel topography since the reflected light does not contain contributions from light reflected from greater depths within the enamel tissue. Multilayered Monte Carlo simulations were also performed to confirm the experimental results.
Short-wavelength 405 nm laser illumination of surface dental enamel using an ultrathin scanning fiber endoscope (SFE)
produced enhanced detail of dental topography. The surfaces of human extracted teeth and artificial erosions were
imaged with 405 nm, 444 nm, 532 nm, or 635 nm illumination lasers. The obtained images were then processed offline
to compensate for any differences in the illumination beam diameters between the different lasers. Scattering and
absorption coefficients for a Monte Carlo model of light propagation in dental enamel for 405 nm were scaled from
published data at 532 nm and 633 nm. The value of the scattering coefficient used in the model was scaled from the
coefficients at 532 nm and 633 nm by the inverse third power of wavelength. Simulations showed that the penetration
depth of short-wavelength illumination is localized close to the enamel surface, while long-wavelength illumination
travels much further and is backscattered from greater depths. Therefore, images obtained using short wavelength laser
are not contaminated by the superposition of light reflected from enamel tissue at greater depths. Hence, the SFE with
short-wavelength illumination may make it possible to visualize surface manifestations of phenomena such as
demineralization, thus better aiding the clinician in the detection of early caries.
Autofluorescence spectra were recorded in vitro from dentin, enamel, and whole teeth. The spectra exhibited a broad peak shifted by about 50 to 75 nm from the excitation wavelength and the shape of the spectra remained similar regardless of the excitation wavelength. The maximum of the autofluorescence spectra also exhibited a red-shift that depended upon the laser excitation wavelength. The amplitude of the red-shifted fluorescence spectra produced by 444 and 532 nm excitation lasers were compared to that produced by a 405 nm excitation laser. It was determined that the autofluorescence amplitude was not proportional to the inverse fourth power of the excitation laser wavelength. Therefore, the red-shifted fluorescence is not compatible with the previously proposed mechanism of Raman scattering. Instead, the mechanism giving rise to the laser-induced dental autofluorescence is explained by the red-edge-excitation effect.
Active Matrix Liquid Crystal Displays (AMLCDs) used in avionics applications require high luminance, high efficacy, and long-life backlights. Currently, fluorescent lamps are the favored light sources for these high performance avionics backlights. Their spectral characteristics and high electrical efficiency are well suited to illuminating AMLCDs used in avionics applications. Fluorescent lamps, however, suffer gradual reduction in luminance output caused by various degradation mechanisms. Korry Electronics Co. recently developed a mathematical model for predicting fluorescent lamp life. The model's basis is the well characterized exponential decay of the phosphor output. The primary luminance degradation mechanism of a fluorescent lamp is related to the arc discharge. Consequently, phosphor depreciation is proportional to the discharge arc power divided by the phosphor surface area. This 'wall loading' is a parameter in the computer model developed to extrapolate long-term luminance performance. Our model predicts a rapidly increasing decay rate of the lamp output as the input power is increased to sustain constant luminance. Eventually, a run-away condition occurs -- lamp arc power must be increased by unrealistically large factors (greater than 5x) to maintain the required luminance output. This condition represents the end of the useful lamp life. The lamp life model requires the definition of several key parameters in order to accurately predict the useful lamp life of an avionics backlight. These important factors include the construction of the lamp, lamp arc power, a decay constant based on the phosphor loading, and the operational profile. Based on the above-mentioned factors, our model approximates the useful lamp life of an avionics backlight using fluorescent lamp technology. Comparisons between calculated and experimental lamp depreciation are presented.
A `slow scan' CCD camera has been adapted for luminance and radiance measurement of displays used in night vision goggle (NVG) compatible aircraft. A video lightmeter offers several advantages compared to conventional test methods including high speed image capture and color coding of the digital image data. The color coding feature facilitates evaluation of the test display uniformity. Numerical values for luminance and infrared radiance are also extracted from the image data.
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