Incident Light Microscopy

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Basically, there are 4 ways to illuminate the observed object

1. Illumination in transmitted light - light passes through the preparation

a. In a bright field
b. In a dark field


2. Illumination in incident - light is reflected from the specimen

a. In a bright field
b. In a dark field

Illumination in incident light in bright field (2a) 

        - It is a system that illuminates an opaque object that is observed from the same side from which it is illuminated.
        - The image is formed on the structure of changes reflectivity of the object.
        - An intact object without a diffusers reflecting structure will provide a field with unevenly distributed brightness.

Illumination in incident light in a dark field (2b) 

        - Light hits the beam obliquely, the beam is reflected outside objective.
        - A perfectly reflective object without structure will create a uniform dark field.

Illumination system in incident light in a bright field

It is used in case of illumination of opaque preparations. Opacity is caused either by high absorption or by the impossibility of preparing a thin enough section. In the case of illumination systems in the bright field, the sample is always illuminated from the same side from which it is observed, i.e. from which the light is reflected into the microscope.


Types of lighting systems


1. Direct lighting
- The preparation is illuminated directly by the source.
- The system shows significant light losses.
2. Condenser
- The source is displayed by a condenser on the object.
- Uneven illumination of the preparation.
3. Vertical Illumination
- The light from the source illuminates the specimen from above through a objective with the help of a semitransmissive mirror or a reflective prism placed eccentrically in the tube.
- Used for higher magnification.
4. Köhler view
- The object is not illuminated directly by the source, but by its image lying in the plane of the condenser aperture. That is, the source is imaged by a collector on the subject main plane of the condenser.
- The source image formed by the collector is imaged by the condenser onto the object.
- The image of the source must fill the entire aperture of father condenser.
- The advantage is the high quality of illumination of the specimen.


Illumination system in incident light in a dark field

Dark Field Method

It is used in the case of observation of small structures, the size of which is below the resolving power of a microscope, working in bright field. The presence of these structures Cana be demonstrated by increasing the contrast when observing in the dark field. The principle of the dark field method consists in the use of scattered light radiation on the inhomogeneities of the preparation structure. We screen off the central rays with a suitable aperture and use only the peripheral rays, which, however, do not enter the lens directly. Therfore, field of view is dark if the specimen is not inserted into the microscope. After inserting the specimen due to reflection, refraction, scattering or diffraction, the light enters the lens and creates an image of the preparation as a glowing structure on a light background - this increases the |contrast of the image.


Types of lighting systems

<Illumination adjustment for the dark field method in incident light is achieved using

a. devices illuminating unilaterally

1. Lamps
- the light rays of the source are concentrated by a collector
- suitable when using weaker lenses
2. Illuminators for oblique lighting
- light is concentrated from the source by the illuminator by reflections from mirror surfaces
- suitable when using stronger lenses
b. devices illuminating from all sides
1. Lieberkühńs Mirror
- The titled mirror surrounds the lens in a ring.
- The preparation is placed on the metal support of the glass plate, which lies on the opening of the cross stage of the microscope.
- The beam passes through the opening of the stage, parallel to the objective, and is reflected onto the specimen using an inclined mirror.
2. Hauser´s construction
- Similar principle.
- However, it eliminates the need for a large opening in the microscope stage.


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References[edit | edit source]

  • HEJTMÁNEK, Milan. Introduction to Light Microscopy :  Intended for post. Faculty of Medicine Univ. Palacký. 3. edition. Olomouc : Palacký University, 1993. ISBN 80-7067-308-7.
  • Navrátil, Rosina a kol.: Medical biophysics. GRADA Publishing, 2010
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