Confocal laser scanning microscopy (CLSM or LSCM) is a valuable tool for obtaining high resolution images and 3-D reconstructions. The key feature of confocal microscopy is its ability to produce blur-free images of thick specimens at various depths. The principle for this special kind of microscopy was developed by Marvin Minsky in 1953, but it took another thirty years and the development of lasers as idealised point sources until confocal microscopy became a standard technique at the end of the eighties.
In a laser scanning confocal microscope a laser beam passes a light source pinhole and is then focused by an objective lens into a small volume element of a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. The dichroic mirror separates the light mixture by reflecting the laser light and allowing only the fluorescent light to pass into the detection region. After passing a pinhole the fluorescent light is detected by a photo-detection device (photomultiplier or avalanche photodiode) transforming the light signal into an electrical one, which is finally evaluated by a computer.
As seen in the figure the detector pinhole obstructs the so called out-of-focus light i.e. fluorescent light not originating from the focal plane of the objecte lens. Light rays from below the focal plane (dashed lines) come to a focus before reaching the detector pinhole, and then they expand out so that most of the rays are physically blocked from reaching the detector by the pinhole. In the same way, light from above the focal plane (dotted lines) is focused behind the detector pinhole, so that most of this light also hits the edges of the pinhole and is not detected. However, all the light from the focal plane (solid lines) is focused at the pinhole and is detected. In this way, out-of-focus information from above and below the focal plane is greatly reduced, which results in blur-free and sharper images compared to conventional microscopy techniques. The detected light originating from an illuminated volume element within the specimen represents one pixel in the resulting image. As the laser scans over the object of interest a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light. The scanning system, which is entered by the excitation light before it is focused onto the specimen, is made up of a reflecting mirror controlled by the computer. This scanning method usually has a low reaction latency and the scan speed can be varied as slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. Furthermore the computer can generate a three-dimensional picture of a specimen by assembling a stack of two-dimensional images from successive focal planes.
In addition, confocal microscopy provides a significant improvement in lateral resolution and the capacity for direct, non-invasive serial optical sectioning of intact, thick living specimens with an absolute minimum of sample preparation. As laser scanning confocal microscopy depends on fluorescence, a sample usually needs to be treated with fluorescent dyes to make things visible. However, the actual dye concentration can be very low so that the disturbance of biological systems is kept to a minimum. Even the tracks of a virus marked with only one single dye molecule can be traced in this way.
As laser scanning confocal microscopy is a scanning imaging technique the way the resolution is obtained might be best explained by comparing it with another scanning technique like scanning tunneling microscopy STM. In STM the image is obtained by scanning with an atomic tip over a conducting surface, while associating a discrete tunnel current with each area element of the surface. If the tip is blunt, i.e. it consists of several atoms, the resolution is bad whereas if the tip is very sharp and made of just one atom atomic resolution is obtained.
In LSCM a fluorescent specimen is illuminated diffraction limited by a laserpoint source, i.e. each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning tip, which is crucial for the obtained resolution, is determined by the diffraction limit of the optical system. This is due to the fact that the image of the scanning laserpoint source is not an infinitely small point but a three-dimensional diffraction pattern. The actual size of this diffraction pattern i.e. the size of the illuminated volume element and thus the obtained resolution depends on the numerical aperture of the objective lens as well as on the wavelength of the used laser light. This can be seen as the classical resolution limit of conventional optical microscopes using a so-called wide-field illumination. However, with confocal microscopy it is even possible to overcome this resolution limit of wide-field illuminating techniques as only light generated in a small volume element is detected at a time. Here it is very important to note, that the effective volume of light generation, is usually smaller than the volume of illumination i.e the diffraction pattern of detectable light creation is sharper and smaller than the diffraction pattern of illumination. In other words, the resolution limit in confocal microscopy depends not only on the probability
of illumination but also on the probability of creating enough detectable photons, so that the actual addressable volume being associated with a generated light intensity is smaller than the illuminated volume. Depending on the fluorescence properties of the used dyes, there is a more or less subtle improvement in lateral resolution compared to conventional microscopes. However, by using light creation processes with much lower probabilities of occurrence such as secondary harmonic generation effects SHG the volume of addressing is reduced to a small region of highest laser illumination intensity resulting in a significant improvement in lateral resolution. Unfortunately, the probability decrease in creation of detectable photons has a bad effect on the signal to noise ratio. This can be compensated either by using more and more sensitive photo-detectors or by increasing the intensity of the illuminating laserpoint source at the risk of destroying the specimen of interest.
As mentioned above, the good axial resolution (between two focal planes) is obtained by using the detector pinhole giving this technique its name. This pinhole would become obsolete, if it were possible to reduce the volume of light
generation to a single point.Image Formation
Resolution Enhancement by the Confocal Principle