In this work, we compare standard wide-field fluorescence lifetime imaging microscopy (FLIM) and structured illumination FLIM (SI-FLIM) as methods for the early detection of oral squamous cell carcinoma (OSCC). Our technique, SI-FLIM, provides depth dependent fluorescence lifetime information of the oral epithelium, isolating the endogenous fluorophore of interest, NADH, from interfering fluorescence generated mainly by collagen in the lamina propria. Male golden Syrian hamsters (Cricetus auratus) were used as the animal model for OSCC. They were treated with a carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), for a twelve-week period by applying the DMBA suspended in mineral oil to their cheek pouches 3 times per week. The progression of OSCC was monitored over a 12-week period with imaging beginning at the 6th week. The cheek pouch with lesions was imaged in a 3x4 grid (twelve total images), with each section of the grid being correlated with histopathological analysis. The NADH fluorescence channel, as a diagnostic indicator, was compared for both SI-FLIM and widefield FLIM. ROC analysis, in the task of distinguishing between mild dysplasia and normal tissue, showed that SI-FLIM (AUC=0.83, se=0.07) may be a better indicator for early cases of mild dysplasia when compared to widefield FLIM (AUC=0.63, se=0.07) with statistical significance.
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.
We present a volumetric imaging method for biological tissue that is free of mechanically scanning components. The optical sectioning in the system is obtained by structured illumination microscopy (SIM) with the depth of focus being varied by the use of an electronic tunable-focus lens (ETL). The performance of the axial scanning mechanism was evaluated and characterized in conjunction with SIM to ensure volumetric images could be recorded and reconstructed without significant losses in optical section thickness and lateral resolution over the full desired scan range. It was demonstrated that sub-cellular image resolutions were obtainable in both microsphere films and in ex vivo oral mucosa, spanning multiple cell layers, without significant losses in image quality. The mechanism proposed here has the ability to be integrated into any wide-field microscopy system to convert it into a three-dimensional imaging platform without the need for axial scanning of the sample or imaging optics. The ability to axially scan independent of mechanical movement also provides the opportunity for the development of endoscopic systems which can create volumetric images of tissue in vivo.
We present a wide-field fluorescence lifetime imaging (FLIM) system with optical sectioning by structured illumination microscopy (SIM). FLIM measurements were made using a time gated ICCD camera in conjunction with a pulsed nitrogen dye laser operating at 450 nm. Intensity images were acquired at multiple time delays from a trigger initiated by a laser pulse to create a wide-field FLIM image, which was then combined with three phase SIM to provide optical sectioning. Such a mechanism has the potential to increase the reliability and accuracy of the FLIM measurements by rejecting background intensity. SIM also provides the opportunity to create volumetric FLIM images with the incorporation of scanning mechanisms for the sample plane. We present multiple embodiments of such a system: one as a free space endoscope and the other as a fiber microendoscope enabled by the introduction of a fiber bundle. Finally, we demonstrate the efficacy of such an imaging system by imaging dyes embedded in a tissue phantom.
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