Technology -
Scanning Laser Confocal Microscope

Many people will remember their experience with microscopes. Even though the object was magnified, certain details always appeared to be fuzzy. At work is a basic principle of optics that prevents both  magnification and the ability to view all the details of a thick specimen. The term used to describe the thickness of the sample that can be viewed in sharp detail is called depth-of-field.  Up to the mid 1980's depth-of-field limited the ability to see objects in detail. This is especially true at high magnifications. Early in the 1980's inexpensive lasers, high speed computers, and a very ingenious microscope design were combined to create the first commercial scanning-laser confocal microscope.

Confocal imaging - what is it?  The basic principle of a confocal microscope is shown below.


  • In the confocal microscope all structures that are out of focus are eliminated  at image formation.
    This is possible because the object is not being illuminated and imaged entirely at the same time, but rather one single point after the other. As shown in the diagram, this is obtained by the arrangement of diaphragms, which act as a point source and as a point detector respectively (solid line). Rays from out-of-focus areas are prevented from being imaged by the final confocal pinhole opening (dashed lines).

  • Using a confocal microscope eliminates out of focus points while retaining the sharp sample areas. Part of the problem for microscopes has been solved. Depth-of-field has been circumvented and sharp images can be viewed, but only for very thin slices. 

What about thick materials? The next important development was high speed inexpensive graphic computers and 3-D software.

  • Using a computer, multiple slices of in-focus images are collected and saved. These slices as a group are called a stack. 

  • Using 3-D software, the stack is reassembled into one sharp image (also called a projection). Take a look and see how this works.

What does a confocal microscope look like?

The Leica TCS NT is a universal Laser Scanning Confocal Microscope System for the bio-medical and materials research environment. The system includes four lasers that can be used simultaneously. The lasers and laser lines include, an Argon (488nm), Krypton (568), RHeNe (633), and an Argon UV (363).

There are two separate microscopes (upright and inverted) that can accommodate the scanning head, which makes the system flexible for various research needs.

What are the advantages and limitations of confocal microscopes?

Major improvements offered by a confocal microscope over the performance of a conventional microscope may be summarized as follows:

  1. Light rays from outside the focal plane will not be

  2. Defocusing does not create blurring, but gradually cuts
    out parts of the object as they move away from the
    focal plane. The practical consequence is that these
    parts become darker and eventually disappear. This
    feature is called optical sectioning.

  3. True, three-dimensional data sets can be recorded.

  4. Scanning the object in x/y-direction as well as in z-direction (along the optical axis) allows viewing the object from all sides.

  5. Due to the small dimension of the illuminating light spot in the focal plane, stray light is minimized.

  6. By image processing, many slices can be superimposed, giving an extended focus image which can only be achieved in conventional microscopy by reduction of the aperture and thus sacrificing resolution.

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