Endomicroscopy
Endomicroscopy is a technique for obtaining histology-like images from inside the human body in real-time, a process known as ‘optical biopsy’. It generally refers to fluorescence confocal microscopy, although multi-photon microscopy and optical coherence tomography have also been adapted for endoscopic use. Commercially available clinical and pre-clinical endomicroscopes can achieve a resolution on the order of a micrometre, have a field-of-view of several hundred μm, and are compatible with fluorophores which are excitable using 488 nm laser light. The main clinical applications are currently in imaging of the tumour margins of the brain and gastro-intestinal tract, particularly for the diagnosis and characterisation of Barrett’s Esophagus, pancreatic cysts and colorectal lesions. A number of pre-clinical and transnational applications have been developed for endomicroscopy as it enables researchers to perform live animal imaging. Major pre-clinical applications are in gastro-intestinal tract, toumour margin detection, uterine complications, ischaemia, live imaging of cartilage and tendon and organoid imaging.
Principles
Conventional, widefield microscopy is generally unsuitable for imaging thick tissue because the images are corrupted by a blurred, out-of-focus background signal. Endomicroscopes achieve optical sectioning using the confocal principle - each image frame is assembled in a point-by-point fashion by scanning a laser spot rapidly over the tissue. In table-top confocal microscopes the scanning is usually performed using bulky galvanometer or resonant scanning mirrors. Endomicroscopes either have a miniaturised scanning head at the distal tip of the imaging probe, or perform the scanning outside of the patient and use an imaging fibre bundle to transfer the scan pattern to the tissue.Single Fibre Endomicroscopes
Single fibre confocal endomicroscopes use the tip of an optical fibre as a spatial filter, enabling miniaturisation of the microscope. 488nm blue laser passes from the source through an optical fibre to a flexible hand-held probe. Optics in the probe focus the laser to a spot in the tissue, exciting fluorescence. Emitted light is captured into the optical fibre and passed through an optical filter to a detector. An image is generated by scanning the focused spot throughout the image plane and compiling the point intensity measurements. The image plane can be translated up and down in the sample, allowing generation of 3D image stacks. Single fibre endomicroscopes have similar resolution of a conventional confocal microscope.Fibre Bundle Endomicroscopes
Fibre bundles were originally developed for use in flexible endoscopes. and have since been adapted for use in endomicroscopy. They consist of a large number of fibre cores inside a single shared cladding, are flexible, and have diameters on the order of a millimetre. In a coherent fibre bundle the relative positions of the cores are maintained along the fibre, meaning that an image projected onto one end of the bundle will be transferred to the other end without scrambling. Therefore, if one end of the bundle is placed at the focus of a table-top confocal microscope, the bundle will act as a flexible extension and allow endoscopic operation.Since only the cores, and not the cladding, transmit light, image processing must be applied to remove the resulting honeycomb-like appearance of the images. Each core essentially acts as an image pixel, and so the spacing between fibre cores limits the resolution. The addition of micro-optics at the distal tip of the bundle allows for magnification and hence higher resolution imaging, but at the cost of reducing the field-of-view.