Oblique illumination

.

With normal bright field illumination, a specimen is evenly illuminated from all sides. When the light that illuminates the specimen is coming from one side, it is called oblique illumination. With oblique illumination, less light is shining directly through the specimen. In stead, the light rays are illuminating the specimen at an angle which gives a relief effect. This illumination technique is very old and is used to increase the visibility of difficult specimens.

With oblique illumination, both contrast and resolution are enhanced. A pseudo 3-dimensional effect is created that gives depth to structures in the specimen thereby increasing visibility.

Oblique illumination can be achieved in many different ways. By simply holding a finger between the light source and condenser, already a kind of oblique lighting is achieved. In principle, any intervention in the light path that results in an uneven illumination of the specimen qualifies as oblique illumination. But some methods are better than others. A proven method for creating oblique illumination is the usage of a darkfield stop or a phase contrast condenser. Also, with an additional lens underneath the condenser, like for example an auxiliary lens, a nice oblique illumination can be created when the lens is shifted.

For the following tests I used a slide of Pleurosigma angulatum and a Zeiss-Winkel achromat 40/0.65. For a 40/0.65 objective, Pleursigma angulatum is a critical object; with insufficient resolution due to incorrect settings, the pore structure of this diatom will not visible. It is therefore a good test specimen to check the resolving power of a 40/0.65 objective. The experiments were performed with a Zeiss Standard GFL using the 0.9 NA flip-top Abbe condenser. The oblique illumination was created with different methods, each time another part of the microscope was shifted. When experimenting with oblique illumination it is important that the field diaphragm and aperture diaphragm are fully opened. Furthermore, the use of a phase-telescope or Bertrand lens is strongly recommended. These tools are used to monitor and record the illumination at the back lens of the objective so that optimal settings can be repeated later. The degree to which a certain part of the microscope is shifted and the height of the condenser have a drastic effect on the results. It is best to experiment with this as much as possible. Figure 1 shows some parts of the Standard GFL that were used to alter the illumination.

Fig.1. Methods to achieve oblique illumination. A: Darkfield stop of 18 mm in diameter in filter holder. B: Shifting the auxiliary lens. C: Shifting the filter holder. D: Tilted condenser top lens.

The following images of Pleurosigma angulatum were made using different settings. The effect of the settings (numbered 1 to 6) on the illumination at the back lens of the objective as seen through a phase-telescope is shown in figure 5.

Fig.2. Pleurosigma angulatum in normal bright field illumination (left, setting 1) and oblique illumination using a darkfield stop in the filter holder (right, setting 2). In the right image, the fine structure of this diatom is much more visible.

Fig.3. Oblique illumination by decentering the condenser (left, setting 3) or by shifting the auxiliary lens under the condenser (right, setting 4).

Fig.4. Oblique illumination by tilting the condenser top lens (left, setting 5) or by positioning the filter holder in the light path (right, setting 6).

Fig. 5. Overview of the different settings. 1: Bright field illumination. 2: Darkfield stop. 3: Decentering the condenser. 4: Auxiliary lens shifted under condenser. 5: Tilted condenser top lens. 6: Filter holder positioned in light path. On the far right of the picture, the illumination at the back lens of the objective is seen, photographed using a phase telescope.

Oblique illumination enhances contrast and resolving power and can be achieved with simple means. There are many different ways to create oblique illumination and each method has it’s own characteristics. I personally find that in the experiments conducted the best contrast and resolving power was obtained by shifting the auxiliary lens (setting 4) or using a darkfield stop in the filter holder (setting 2). In my opinion, these two methods proved to be best in resolving the structure of Pleurosigma angulatum. On the other hand, decentering the condenser (setting 3) produced the least color artifacts and a nearly gradient-free oblique illumination was obtained.

Depth of field

The depth of field can be increased considerably with oblique illumination. It gives a more spatial impression of the object than normal brightfield illumination and the visibility of some details will be enhanced. If you look at photos taken with oblique illumination, there may be a sudden reversal of the relief; cavities suddenly look like bulges. This is an optical illusion. It is good practice to always take a picture in brightfield and compare it with the image taken with oblique lighting.

Arachnoidiscus ehrenbergii fotographed in brightfield (left) and oblique illumination (right). Objective: Zeiss-Winkel 25/0.45.