We describe the design and operation of a multispectral confocal microendoscope. This fiber-based fluorescence imaging system consists of a slit-scan confocal microscope coupled to an imaging catheter that is designed to be minimally invasive and allow for cellular level imaging in vivo. The system can operate in two imaging modes. The grayscale mode of operation provides high resolution real-time in vivo images showing the intensity of fluorescent signal from the specimen. The multispectral mode of operation uses a prism as a dispersive element to collect a full multispectral image of the fluorescence emission. The instrument can switch back and forth nearly instantaneously between the two imaging modes (less than half a second). In the current configuration, the multispectral confocal microendoscope achieves 3-μm lateral resolution and 30-μm axial resolution. The system records light from 500 to 750 nm, and the minimum resolvable wavelength difference varies from 2.9 to 8.3 nm over this spectral range. Grayscale and multispectral imaging results from ex-vivo human tissues and small animal tissues are presented.
Optical biopsy facilitates in vivo disease diagnoses by providing a real-time in situ view of tissue in a clinical setting. Fluorescence confocal microendoscopy and optical coherence tomography (OCT) are two methods that have demonstrated significant potential in this context. These techniques provide complementary viewpoints. The high resolution and contrast associated with confocal systems allow en face visualization of sub-cellular details and cellular organization within a thin layer of biological tissue. OCT provides cross-sectional images showing the tissue micro-architecture to a depth beyond the reach of confocal systems. We present a novel design for a bench-top imaging system that incorporates both confocal and OCT modalities in the same optical train allowing the potential for rapid switching between the two imaging techniques. Preliminary results using simple phantoms show that it is possible to realize both confocal microendoscopy and OCT through a fiber bundle based imaging system.
Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.
We present an ultrahigh resolution spectral-domain optical coherence tomography imaging system using a broadband superluminescent diode light source emitting at a center wavelength of 1.3 µm. The light source consists of two spectrally shifted superluminescent diodes that are coupled together into a single mode fiber. The effective emission power spectrum has a full width at half maximum of 200 nm and the source output power is 10 mW. The imaging system has an axial resolution of 3.9 µm in air (< 3.0 µm in biological tissue), and a lateral resolution of 6.5 µm. The sensitivity and the maximum line rate are 95 dB and 46 kHz, respectively. Images of an infrared viewing card and a cornea from human eye suffering from glaucoma showing Schlemm's canal are presented to illustrate the performance of the system.
A theoretical analysis of the use of a fiber bundle in spectral-domain optical coherence tomography (OCT) systems is presented. The fiber bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the OCT data. However, the multimode characteristic of the fibers in the fiber bundle affects the depth sensitivity of the imaging system. A description of light interference in a multimode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.
Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.
We present a modified multi-spectral configuration of a slit-scanning confocal microendoscope that provides higher spectral resolution in a fully automated interface. Tissue fluorescence signal is directed through a dispersive element that spreads the spectral information across the CCD camera mapping spectral information perpendicular to the confocal slit. The dispersive element may be chosen to meet the specific requirements defined by the user. Our current system uses a BK7 prism with a 45° wedge angle and a 20mm diameter clear aperture. The prism is shifted from the optical axis allowing automated switching from grayscale (beam on-axis) to multi-spectral (beam off-axis) imaging by tilting a computer controlled mirror. The system records over a spectral range of 450nm to 750nm. The minimum resolvable wavelength difference varies from 2.1nm to 8.3nm over the spectral range. The lateral and axial resolution of the system is approximately 3µm by 30µm, respectively, and is the same for both grayscale and multi-spectral imaging modes. Multi-spectral imaging results from ex-vivo tissues are presented.
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