Microparticle characterization via confocal microscopy  (Page 2/4)

How confocal microscopy is different from optical microscopy

A microscope’s lenses project the sample plane onto an image plane. An image can be formed at many image planes; however, we only consider one of these planes to be the ‘focal plane’ (when the sample image is in focus). When a pinhole screen in placed at the image focal point, it allows in-focus light to pass while effectively blocking light from out-of-focus locations [link] . This pinhole is placed at the conjugate image plane to the focal plane, thus the name "confocal". The size of this pinhole determines the depth-of-focus; a bigger pinhole collects light from a larger volume. The pinhole can only practically be made as small as approximately the radius of the Airy disk, which is the best possible light spot from a circular aperture [link] , because beyond that more signal is blocked resulting in a decreased signal-to-noise ratio.

To further reduce the effect of scattering due to light from other parts of the sample, the sample is only illuminated at a tiny point through the use of a pinhole in front of the light source. This greatly reduces the interference of scattered light from other parts of the sample. The combination of a pinhole in front of both the light source and detector is what makes confocal unique.

Parts of a confocal microscope

A simple confocal microscope generally consists of a laser, pinhole aperture, dichromatic mirror, scanning mirrors, microscope objectives, a photomultiplier tube, and computing software used to reconstruct the image [link] . Because a relatively small volume of the sample is being illuminated at any given time, a very bright light source must be used to produce a detectable signal. Early confocal microscopes used zirconium arc lamps, but recent advances in laser technology have made lasers in the UV-visible and infrared more stable and affordable. A laser allows for a monochromatic (narrow wavelength range) light source that can be used to selectively excite fluorophores to emit photons of a different wavelength. Sometimes filters are used to further screen for single wavelengths.

The light passes through a dichromatic (or "dichroic") mirror [link] which allows light with a higher wavelength (from the laser) to pass but reflects light of a lower wavelength (from the sample) to the detector. This allows the light to travel the same path through the majority of the instrument, and eliminates signal due to reflection of the incident light.