The Principle & Applications of Confocal Imaging

Confocal imaging (CI) is based on illuminating a single point on the sample and collecting scattered light from the same point of the sample. The illumination point on the sample is the image of the illumination pinhole and is imaged into the detector pinhole, hence making both pinholes and the illuminated spot on the sample optically conjugated. In this manner, stray light from points outside the collection volume is effectively filtered, enhancing contrast, specifically for scattering media, for instance, biological tissues. Scanning the position of this point on the sample and acquiring the signal from each position creates a lateral image of the sample. For axial cross-sectional imaging, the focal point on the sample is axially moved while acquiring the signal from each position. Combining both lateral and axial scans provides a perpendicular cross-sectional image of the tissue. The nature of the signal from the illuminated point depends on the specific embodiment. Usually backscattered or fluorescent signals are often used for confocal imaging; but other signals have also been collected and proven clinically helpful.

The source/illumination pinhole and detector pinhole contribute to depth sectioning ability. The source pinhole causes the illuminating irradiance to be strongly peaked at the image formed by the objective and to fall off rapidly both transversely and along the line of sight (depth). Thus, there is, extremely little light available to illuminate out of focus volumes. This means that, these volumes will not significantly mask the image of the more brightly illuminated focal region. The detector pin functions in a similar fashion. It is more efficient in gathering the radiation from the volume at the focal point, and its efficiency falls off rapidly with distance, both transversely and in depth. Both pinholes, work to ensure that the totality of the out of focus signal is strongly rejected.

Confocal imaging provides improved lateral and axial resolutions and improved rejection of light from the out of focus tissue. Thus, confocal imaging can achieve a resolution sufficient for imaging cells to depths of several hundreds of microns. At these depths, confocal imaging has been particularly useful in imaging the epithelium of a number of organs, including internal organs via endoscopic access.

Fiber-optical confocal microscope is a type of confocal imaging microscope, where the light source is not a point source, but the tip of an optical fiber, and the signal is collected by another optical fiber that delivers the signal to a detector. This makes the confocal imaging system compact and thus convenient in many biomedical applications.

Applications of Confocal Imaging

Confocal imaging system is used for example in diagnostics of intraepithelial neoplasia and cancer of the colon.

Also read:

• Atomic Force Microscopy (AFM)
• Optical Tomographic Imaging
• Radionuclide Imaging Techniques
• X-ray Imaging