Confocal laser scanning microscopy

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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. Images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece. 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 for confocal microscopy to become a standard technique toward the end of the 1980s.

1 Image formation
2 Resolution enhancement by the confocal principle
3 Uses
4 See also
5 External links

[edit] Image formation

In a laser scanning confocal microscope a laser beam passes a light source aperture and then is focused by an objective lens into a small (ideally diffraction-limited) focal volume within 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. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole the fluorescent light is detected by a photo-detection device (photomultiplier tube (PMT) or avalanche photodiode) transforming the light signal into an electrical one which is recorded by a computer.

Principle of a confocal microscope.

The detector aperture obstructs the light that is not coming from the focal point, as shown by the dotted grey line in the image. The out-of-focus points are thus suppressed: most of their returning light is blocked by the pinhole. This results in sharper images compared to conventional fluoresence microscopy techniques and permits one to obtain images of various z axis planes (z-stacks) of the sample.

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 plane 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 beam is scanned across the sample in the horizontal plane using one or more (servo-controlled) oscillating mirrors. This scanning method usually has a low reaction latency and the scan speed can be varied. Slower scans provide a better signal to noise ratio resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these 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. Some instruments are capable of tracking single fluorescent molecules. Additionally transgenic techniques can create organisms which produce their own fluorescent chimeric molecules. (such as a fusion of GFP, Green fluorescent protein with the protein of interest).

[edit] Resolution enhancement by the confocal principle

Laser scanning confocal microscopy (LSCM) is a scanning imaging technique in which the resolution obtained is best explained by comparing it with another scanning technique like Scanning electron microscope (SEM). Not to be confused with phonograph-like imaging—AFM or STM, for example, where the image is obtained by scanning with an atomic tip over a conducting surface.

In LSCM a fluorescent specimen is illuminated by a point laser source, and each volume element is associated with a discrete fluorescence intensity. Here, the size of the scanning volume is determined by the diffraction limit of the optical system. This is due to the fact that the image of the scanning laser is not an infinitely small point but a three-dimensional diffraction pattern. The size of this diffraction pattern and the focal volume it defines is controlled by the numerical aperture of the system's objective lens and the wavelength of the laser used. This can be seen as the classical resolution limit of conventional optical microscopes using 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; that is, 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 second harmonic generation (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 by using more sensitive photo-detectors or by increasing the intensity of the illuminating laser point source. Increasing the intensity of illumination latter risks excessive bleaching or other damage to the specimen of interest, especially for experiments in which comparison of fluorescence brightness is required.

[edit] Uses

Confocal microscopy is clinically used in evaluation of various eye diseases. It is particularly useful for imaging, qualitative analysis and quantitafication of endothelial cells of the cornea. It is used for localising and identifying presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy.

[edit] See also

Two-photon excitation microscopy : Although they employ a related technology (both are laser scanning microscopes), multiphoton fluorescence microscopes are not confocal microscopes. The term confocal arises from the presence of a diaphragm in the conjugated focal plane (confocal). This diaphragm is usually absent in multiphoton microscopes.
Total internal reflection fluorescence microscope
Fluorescence microscope
STED microscopy