ABSTRACT
OBJECTIVE: Structured illumination microscopy is an inexpensive alternative to confocal microscopy that allows optical sectioning at a sub-cellular resolution. However, its application in imaging biological tissue has been limited by inadequate contrast present in them especially in reflectance imaging. Novel, optically active contrast agents like gold nanoparticles and quantum dots targeted against biomarkers of cancer can be integrated with structured illumination to image both the morphological and biochemical changes associated with epithelial pre-cancers. METHODS: We modified the optical path of a widefield microscope to implement structured illumination both in reflectance and fluorescence modes. For imaging, we used 25-nm-diameter gold nanoparticles and CdSe quantum dots for reflectance and fluorescence imaging, respectively, to label three-dimensional tissue constructs of SiHa cervical cancer cells. Contrast agents were targeted against the epidermal growth factor receptor (EGFR) using an anti-EGFR monoclonal antibody. Agents targeted with a non-specific IgG antibody served as a control to monitor non-specific labeling. RESULTS: Our result shows that optically sectioned images taken with structured illumination are very comparable to those obtained using confocal microscopy. Moreover, images of three-dimensional cultures stained with the anti-EGFR agents show significantly more image intensity than those stained with the IgG targeted control. CONCLUSION: Our findings suggest that the combination of novel optical contrast agents and structured illumination can differentiate neoplastic cells which overexpress EGFR from normal cells in intact tissue. Combining structured illumination microscopy with novel contrast agents can potentially provide a powerful and inexpensive tool to aid in the detection of cervical pre-cancers.
Subject(s)
Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Precancerous Conditions/diagnosis , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibody Specificity , Cell Line, Tumor , Contrast Media , ErbB Receptors/immunology , ErbB Receptors/metabolism , Female , Fluorescent Dyes/metabolism , Humans , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Quantum Dots , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/metabolism , Uterine Cervical Dysplasia/pathologyABSTRACT
A ball lens coupled fiber-optic probe design is described for depth-resolved measurements of the fluorescence and reflectance properties of epithelial tissue. A reflectance target, fluorescence targets, and a two-layer tissue phantom consisting of fluorescent microspheres suspended in collagen are used to characterize the performance of the probe. Localization of the signal to within 300 microm of the probe tip is observed by use of reflectance and fluorescence targets in air. Differential enhancement of the fluorescence signal from the top layer of the two-layer tissue phantom is observed.
Subject(s)
Epithelium/physiology , Epithelium/ultrastructure , Fiber Optic Technology/instrumentation , Lenses , Microscopy, Fluorescence/instrumentation , Spectrometry, Fluorescence/instrumentation , Tomography, Optical/instrumentation , Animals , Equipment Design , Equipment Failure Analysis , Fiber Optic Technology/methods , Humans , Microscopy, Fluorescence/methods , Optical Fibers , Phantoms, Imaging , Spectrometry, Fluorescence/methods , Tomography, Optical/methods , TransducersABSTRACT
Structured-illumination microscopy delivers confocal-imaging capabilities and may be used for optical sectioning in bio-imaging applications. However, previous structured-illumination implementations are not capable of imaging molecular changes within highly scattering, biological samples in reflectance mode. Here, we present two advances which enable successful structured illumination reflectance microscopy to image molecular changes in epithelial tissue phantoms. First, we present the sine approximation algorithm to improve the ability to reconstruct the in-focus plane when the out-of-focus light is much greater in magnitude. We characterize the dependencies of this algorithm on phase step error, random noise and backscattered out-of-focus contributions. Second, we utilize a molecular-specific reflectance contrast agent based on gold nanoparticles to label disease-related biomarkers and increase the signal and signal-to-noise ratio (SNR) in structured illumination microscopy of biological tissue. Imaging results for multi-layer epithelial cell phantoms with optical properties characteristic of normal and cancerous tissue labeled with nanoparticles targeted against the epidermal growth factor receptor (EGFR) are presented. Structured illumination images reconstructed with the sine approximation algorithm compare favorably to those obtained with a standard confocal microscope; this new technique can be implemented in simple and small imaging platforms for future clinical studies.
