Confocal Microscopy Technique

In conventional microscopy, the specimen is usually mounted on a glass slide and then a coverslip placed to sandwich the specimen between it and the slide. For biological samples, the tissue is often stained to visualize specific cell components and then embedded in paraffin wax. This destroys cell function; nevertheless, there are techniques, case in point, using light polarization properties for visualizing fresh tissues or cell suspensions without the use of stains or embedding, which allow live-cell imaging.

Since, in conventional microscopy, the specimen is illuminated by focusing a light via a condenser lens, there is no way to avoid shadowy images of structures above and below the focal plane from appearing in the viewed image. On the other hand, in confocal microscopy technique, light from above or below the focal plane doesn’t play a role in image formation. This implies that in forming an image of an array of cells, only those structures in the focal plane will be imaged. The rest will not give rise to an out-of-focus blur seen in conventional microscopy.  This achieved by employing an optical device for preventing the rays of light emanating from cones A & B (Refer to the figure 1.0 below) from forming the final image. The most appropriate way of doing this is to use pinholes for both the illuminating beam and the reflected beam (which carries the image information), so that only information from the focal plane (as shown in the figure below, the focal spot), will reach the detector which forms the image.

In the example illustrated in the figure above, the image is formed by scanning the focal spot in a raster pattern such as a TV image over the focal plane. This detector is normally a photomultiplier tube (PMT) or a charge-coupled device (CCD) similar to that found in digital cameras. Hence, a digital image of light intensity in 512 x 512 or more pixels.

Confocal Laser Scanning Microscopy (CLSM)

Confocal laser scanning microscopy (CLSM) also referred to as laser scanning confocal microscopy (LSCM) employs a laser beam as a light source, because the monochromatic lines in a laser source (such as an argon-ion laser which has lines at 351, 454.6, 457.9, 465.8, 476.5, 488.0, 496.5, 501.7, 514.5 and 528.7 nm, but with the strongest emissions at 488 nm and 514.5 nm) can be used to excite specific tissues dyes. The information recorded by the detector is therefore usually fluorescent amplitude rather than reflectance; however the latter has been employed in the studies of silver particles in nerves.

The focal plane can actually be less than a micron thick. If the focal plane is gradually moved through the cell i.e. by moving the stage on which the specimen is placed toward the objective, a stack of two-dimensional image ‘slices’ can be taken. A computer program can be used to render these 2D images as 3D Shape, which can be rotated to give a very clear impression of cell morphology.

Since the focal spot is scanned across each focal plane, the acquisition time for one frame has to be taken into account. In advanced CLSM systems, each image can be obtained in 0.1 s, so a stack of 10 images would take 1 s, although, this depends on the degree of fluorescent intensity. Most often than not, several frames have to be taken to provide an acceptable image. Hence, cell processes which take place on a milliseconds timescale normally cannot be imaged; however there are special techniques to permit this.

Also read:

• Atomic Force Microscopy (AFM)
• The Principle & Applications of Confocal Imaging
• The Principles of Microbial Biosensors
• The Role of Biomedical Analysis in Medical Diagnosis