What is confocal microscopy?
Confocal microscopy is an optical sectioning technique that employs spatial filtering to produce images that are free from out-of-focus blur. The technique commonly uses visible wavelength lasers as light sources and confocal apertures (or "pinholes") in the detection path. Confocal microscopy has gained in popularity in recent years, due in large part to how easy it is to obtain high quality images from samples prepared for conventional optical microscopy.

How does it work?
In conventional optical microscopy, the sample is completely illuminated by the excited light, so all of the sample is fluorescing at the same time, with the highest intensity of the light at the focal point of the lens. This conventional method contributes to a background haze in the resulting image.

Confocal microscopy involves scanning one or more focused beams of light (e.g., laser light) across a sample to illuminate it. Optical sectioning is achieved by incorporating a pinhole into the light collection path. Because the focal point of the objective lens of the microscope forms an image where the pinhole is, these two points are known as conjugate points, and the pinhole becomes a confocal pinhole. Only the in-focus light is detected and passes through the pinhole, while light from outside the focal region is blocked out. A detector (e.g., a photomultiplier tube) measures the light that passes through the pinhole.

Confocal microscopy allows only one point in the sample to be focused on at a time. However, users of this method can construct an image of the complete sample by scanning a laser beam over the entire focal plane and mechanically moving the sample to change the depth of the optical plane through the sample. Users can then reconstruct the image to provide a 3D representation of the sample with all planes in focus.

What are its advantages?
Confocal microscopy offers a number of advantages over conventional optical microscopy:

•• ability to generate in-focus 3D images of microscope samples
•• small depth of field
•• accurate measurements between 2D and 3D space
•• ability to collect serial optical sections from thick samples

What's Confocal microscopy used for?
•• semiconductor UV metrology
•• high resolution analysis of semiconductor defects
•• biomedical fluorescence observations
•• tissue research for cytology