Rodrigo Cuenca

Optical axial scanning in confocal microscopy using an electrically tunable lens

This paper presents the use and characterization of an electrically focus tunable lens to perform axial scanning in a confocal microscope. Lateral and axial resolution are characterized over a >250 µm axial scan range. Confocal microscopy using optical axial scanning is demonstrated in epithelial tissue and compared to traditional stage scanning. By enabling rapid axial scanning, minimizing motion artifacts, and reducing mechanical complexity, this technique has potential to enhance in vivo three-dimensional imaging in confocal endomicroscopy.

Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer

Abstract. 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.

Handheld multispectral fluorescence lifetime imaging system for in vivo applications

There is an increasing interest in the application of fluorescence lifetime imaging (FLIM) for medical diagnosis. Central to the clinical translation of FLIM technology is the development of compact and high-speed clinically compatible systems. We present a handheld probe design consisting of a small maneuverable box fitted with a rigid endoscope, capable of continuous lifetime imaging at multiple emission bands simultaneously. The system was characterized using standard fluorescent dyes. The performance was then further demonstrated by imaging a hamster cheek pouch in vivo, and oral mucosa tissue both ex vivo and in vivo, all using safe and permissible exposure levels. Such a design can greatly facilitate the evaluation of FLIM for oral cancer imaging in vivo.

Reflectance confocal endomicroscope with optical axial scanning for in vivo imaging of the oral mucosa

This paper presents the design and evaluation of a reflectance confocal laser endomicroscope using a miniature objective lens within a rigid probe in conjunction with an electrically tunable lens for axial scanning. The miniature lens was characterized alone as well as in the endoscope across a 200 µm axial scan range using the tunable lens. The ability of the confocal endoscope to probe the human oral cavity is demonstrated by imaging of the oral mucosa in vivo. The results indicate that reflectance confocal endomicroscopy has the potential to be used in a clinical setting and guide diagnostic evaluation of biological tissue.

Estimation of the number of fluorescent end-members for quantitative analysis of multispectral FLIM data

Multispectral fluorescence lifetime imaging (m-FLIM) can potentially allow identifying the endogenous fluorophores present in biological tissue. Quantitative description of such data requires estimating the number of components in the sample, their characteristic fluorescent decays, and their relative contributions or abundances. Unfortunately, this inverse problem usually requires prior knowledge about the data, which is seldom available in biomedical applications. This work presents a new methodology to estimate the number of potential endogenous fluorophores present in biological tissue samples from time-domain m-FLIM data. Furthermore, a completely blind linear unmixing algorithm is proposed. The method was validated using both synthetic and experimental m-FLIM data. The experimental m-FLIM data include in-vivo measurements from healthy and cancerous hamster cheek-pouch epithelial tissue, and ex-vivo measurements from human coronary atherosclerotic plaques. The analysis of m-FLIM data from in-vivo hamster oral mucosa identified healthy from precancerous lesions, based on the relative concentration of their characteristic fluorophores. The algorithm also provided a better description of atherosclerotic plaques in term of their endogenous fluorophores. These results demonstrate the potential of this methodology to provide quantitative description of tissue biochemical composition.

A novel multimodal optical imaging system for early detection of oral cancer

OBJECTIVES Several imaging techniques have been advocated as clinical adjuncts to improve identification of suspicious oral lesions. However, these have not yet shown superior sensitivity or specificity over conventional oral examination techniques. We developed a multimodal, multi-scale optical imaging system that combines macroscopic biochemical imaging of fluorescence lifetime imaging with subcellular morphologic imaging of reflectance confocal microscopy for early detection of oral cancer. We tested our system on excised human oral tissues. STUDY DESIGN In total, 4 tissue specimens were imaged. These specimens were diagnosed as either clinically normal, oral lichen planus, gingival hyperplasia, or superficially invasive squamous cell carcinoma. The optical and fluorescence lifetime properties of each specimen were recorded. RESULTS Both quantitative and qualitative differences among normal, benign, and squamous cell carcinoma lesions can be resolved with fluorescence lifetime imaging reflectance confocal microscopy. The results demonstrate that an integrated approach based on these two methods can potentially enable rapid screening and evaluation of large areas of oral epithelial tissue. CONCLUSIONS Early results from ongoing studies of imaging human oral cavity illustrate the synergistic combination of the 2 modalities. An adjunct device based on such optical characterization of oral mucosa can potentially be used to detect oral carcinogenesis in early stages.

Handheld tunable focus confocal microscope utilizing a double-clad fiber coupler for in vivo imaging of oral epithelium

Abstract. A reflectance confocal endomicroscope with double-clad fiber coupler and electrically tunable focus lens is applied to imaging of the oral mucosa. The instrument is designed to be lightweight and robust for clinical use. The tunable lens allows axial scanning through >250  μm in the epithelium when the probe tip is placed in contact with tissue. Images are acquired at 6.6 frames per second with a field of view diameter up to 850  μm. In vivo imaging of a wide range of normal sites in the oral cavity demonstrates the accessibility of the handheld probe. In vivo imaging of clinical lesions diagnosed as inflammation and dysplasia illustrates the ability of reflectance confocal endomicroscopy to image cellular changes associated with pathology.

Machine learning methods for fluorescence lifetime imaging (FLIM) based automated detection of early stage oral cancer and dysplasia (Conference Presentation)

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.

Objective Detection of Oral Carcinoma with Multispectral Fluorescence Lifetime Imaging In Vivo

Successful early detection and demarcation of oral carcinoma can greatly impact the associated morbidity and mortality rates. Current methods for detection of oral cancer include comprehensive visual examination of the oral cavity, typically followed by tissue biopsy. A noninvasive means to guide the clinician in making a more objective and informed decision toward tissue biopsy can potentially improve the diagnostic yield of this process. To this end, we investigate the potential of fluorescence lifetime imaging (FLIM) for objective detection of oral carcinoma in the hamster cheek pouch model of oral carcinogenesis in vivo. We report that systematically selected FLIM features can differentiate between low‐risk (normal, benign and low‐grade dysplasia) and high‐risk (high‐grade dysplasia and cancer) oral lesions with sensitivity and specificity of 87.26% and 93.96%, respectively. We also show the ability of FLIM to generate “disease” maps of the tissue which can be used to evaluate relative risk of neoplasia. The results demonstrate the potential of multispectral FLIM with objective image analysis as a noninvasive tool to guide comprehensive oral examination.

Handheld optical system for fast and accurate in vivo fluorescence lifetime imaging (FLIM) (Conference Presentation)

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.