Laser Scan Confocal Microscopy

Confocal microscopy is an imaging technique used to increase image contrast and to reconstruct three-dimensional images by using a pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.

Confocal microscopy provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution. With Confocal microscopy, it is even possible to improve on the resolution limit of wide-field illumination techniques because the Confocal aperture can be closed down to eliminate higher orders of the diffraction pattern. The principle of Confocal imaging was patented by Marvin Minsky in 1957.

Basic concept

In a Fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a Photodetector. But the Confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector .This eliminates the out-of-focus light. Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. lens, and also by the optical properties of the specimen and the ambient index of refraction

Confocal Microscopes

There are three types of Confocal microscopes. These are

1. Confocal laser scanning microscopes

Confocal laser scanning microscopy yields better image quality. Confocal microscopes can achieve video rate imaging—a desirable feature for dynamic observations such as lives cell imaging.

2. Spinning-disk Confocal microscopes. Confocal laser scanning microscopy has now been improved to provide better than video rate (60 frames/second) imaging by using MEMS based scanning mirrors.

3. Programmable Array Microscopes -PAM

Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images.

Key feature of Confocal microscopy are

1. Its ability to produce in-focus images of thick specimens, a process known as optical sectioning.

2. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects.

Image formation

Image formation in Confocal Microscope occurs in the following sequence

1. A laser beam passes through a light source aperture and then is focused by an objective lens into a small focal volume within a fluorescent specimen.

2. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens.

3. 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.

4. After passing a pinhole, the fluorescent light is detected by a photodetection device (a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer.

5. The detector aperture obstructs the light that is not coming from the focal point. The out-of-focus light is suppressed: most of their returning light is blocked by the pinhole, resulting in sharper images than those from conventional fluorescence microscopy techniques, and permits one to obtain images of various z axis planes (also known as z stacks) of the sample.

6. 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, whereas the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light.

7. The beam is scanned across the sample in the horizontal plane by using one or more (servo controlled) oscillating mirrors. 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.

8. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.


1. Confocal Laser Scan Microscope is widely-used in numerous biological applications from cell biology and genetics to microbiology and developmental biology.

2. Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells of the cornea.

3. It is used for localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis.

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Laser Scan Confocal Microscope

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