Principles of confocal microscopy

Confocal microscopy provides better spatial resolution than conventional fluorescence microscopy, as the images are not contaminated by light scattering from other focal planes. A low-powered laser is focused onto a single point in a defined microscopic field of view, and the same lens is used as both the condenser and objective folding optical path. The point of illumination thus coincides with the point of detection within the specimen. Light emanating from that point is focused through a pin-hole to a detector, and light emanating from outside the illuminated spot is rejected. The illumination and detection systems are in the same focal plane and are termed “confocal”. All detected signals from the illuminated spot are captured and measured. The gray-scale image created is an optical section representing one focal plane within the examined specimen. The image of a scanned region can be constructed and digitized by measuring the light returning to the detector from successive points. Single points are typically scanned in a raster pattern. Series of confocal images within successive planes can be used to observe fine cellular or subcellular structures, and three-dimensional structures in the specimen can be imaged. Confocal microscopy has become a standard method for molecular imaging in basic research, in conjunction with fluorescence labeling techniques, selectively imaging the position of specific proteins at distinct cellular locations. However, confocal microscopy has so far mainly been carried out on a microscope stage on the laboratory bench.

		Scheme of confocal laser endomicroscopy

Recently, a unique miniaturized design was developed, using a single optical-mode fiber acting as both the illumination point source and the detection pinhole (Optiscan Pty. Ltd., Notting Hill, Victoria, Australia). In addition to standard video imaging, the confocal laser microscope, integrated into the distal tip of a conventional video endoscope (Pentax EC-3870CIFK; Pentax, Tokyo, Japan), also allows confocal microscopy of the mucosal layer at high resolution (lateral resolution 0.7 µm).