For brightfield illumination to be effective, there needs to be a variation in opacity across the object. Without this variation, the illumination creates a dark blur around the object, resulting in an image with relative contrast between the object’s parts and the light source. Typically, brightfield illumination allows clear visualization of each part of the object unless it is extremely transparent. In cases where transparency hinders feature distinction, darkfield illumination becomes useful.
Darkfield illumination directs light rays obliquely onto the object, avoiding direct entry into the objective. Despite this oblique angle, the rays still illuminate the object plane. The resulting darkfield illumination image achieves high contrast between the transparent object and the light source. In a darkfield setup, a light source forms an inverted cone of light that blocks central rays but allows oblique rays to illuminate the object (see Figure 3). This design effectively forces light to illuminate the object without entering the optical system, making darkfield illumination particularly suitable for transparent objects. In contrast, no rays are blocked in a brightfield illumination setup.Epi-illumination, a third form of illumination employed in microscopy, generates light from above the objective. This setup replaces the need for a Koehler illumination configuration, as both the objective and the epi-illumination source contribute to the illumination process. The compact structure of epi-illumination is a significant advantage, as the objective serves as a primary source for a considerable portion of the illumination. Figure 4 provides a depiction of a frequently used epi-illumination setup, particularly common in fluorescence applications.
Key Concepts and Specifications
The majority of microscope objective specifications are conveniently displayed on the objective’s body, including information such as the objective design/standard, magnification, numerical aperture, working distance, lens to image distance, and cover slip thickness correction. Refer to Figure 5 for guidance on interpreting microscope objective specifications. This direct placement of specifications on the objective facilitates a clear understanding of its characteristics, a crucial aspect when integrating multiple objectives into an application. Any additional specifications, like focal length, field of view (FOV), and design wavelength, can be readily calculated or obtained from the vendor or manufacturer’s provided specifications.- Numerical Aperture (NA): NA is the measure of its capability to gather light and to resolve fine specimen details at a fixed object. A lens with a high NA collects more light and can resolve finer specimen details at a fixed distance. These are the elements that determine resolution, depth of focus, and image brightness. The larger the numerical aperture, the higher the resolution and the brighter the image can be observed. The higher the magnification of the objective lens, the larger the numerical aperture.
- Cover Glass: Objectives are usually corrected for a specific cover glass thickness, with 0.17 millimeters being the standard. The thickness of the cover glass is numerically marked on the objective lens. There are three types: one for cover glass specimens, one for non-cover specimens, and one for both cover glass specimens and no cover glass specimens.
- Immersion Medium: The main purpose of using different types of immersion medium is to minimize the refractive index between the objective and the sample. It is crucial to use the correct medium, such as water, oil, or air/dry, as specified by the objective.
- Working Distance: The distance between the front end of the microscope objective and the surface of the specimen at which the sharpest focus is achieved. Proper positioning is important to obtain a good image at the specified magnification. The distance from the tip of the objective lens to the specimen surface when focused. The larger the numerical aperture of the objective lens, the shorter the working distance.
- Parfocal Length: The distance from the mounting plane of the objective to the sample plane.
- Working Wavelength(s): Objectives are corrected for specific wavelengths, with shorter wavelengths yielding higher resolution.