Leonard Y. Nelson

Mitigating fluorescence spectral overlap in wide-field endoscopic imaging.

Abstract. 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.

Target-to-background enhancement in multispectral endoscopy with background autofluorescence mitigation for quantitative molecular imaging

Abstract. 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.

Red-shifted fluorescence of sound dental hard tissue

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.

Color-matched and fluorescence-labeled esophagus phantom and its applications

Abstract. 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.

Trimodal detection of early childhood caries using laser light scanning and fluorescence spectroscopy: clinical prototype

Abstract. 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.

Spectrally enhanced imaging of occlusal surfaces and artificial shallow enamel erosions with a scanning fiber endoscope

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.

Scanning Fiber Endoscope with multiple fluorescence-reflectance imaging channels for guiding biopsy

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.

Optical Measure of Enamel Health: Ability to Triage High Risk Children in Communities without Dental Practitioners

As prevalence of dental caries in children rises worldwide, there is an increasing need for a safe, easy to use and cost-effective technique to detect and identify childhood caries at an early stage where remineralization of the tooth is possible and damage may be reversed. We have developed a simple and robust autofluorescence (AF) laboratory device that uses the dental AF spectra from 405 nm and 532 nm laser excitation and computes a ratio of the integrated areas of the spectra. Ten human extracted teeth with early stage natural caries lesions and an additional 8 human extracted teeth with artificially created erosion lesions were used for the study. The 405/532 nm AF ratio was obtained from healthy as well as unhealthy enamel regions for all teeth. A clear distinction between the ratios for healthy enamel and unhealthy enamel was seen. A percent change in 405/532 nm AF ratio of 62% was seen between natural white spot lesions and healthy enamel, with progressively more severe lesions leading to greater percent changes in AF ratios. The 405/532 nm AF ratio is a promising technique that may be used to detect the presence of early stage dental caries and triage high risk children. A cost effective clinical device can be developed which utilizes the proposed technique to screen children in underserved communities.

Spectrally enhanced image resolution of tooth enamel surfaces

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.

Real-time porphyrin detection in plaque and caries: a case study

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.