Components of a microscope
A microscope is an optical device designed to magnify the image of an object, enabling details indiscernible to the human eye to be differentiated. A microscope may project the image onto the human eye or onto a camera or video device.
Microscopes are usually complex assemblies that include an array of lenses, filters, polarizers, and beamsplitters. Illumination is arranged to provide enough light for a clear image, and sensors are used to ‘see’ the object.
Although today’s microscopes are usually far more powerful than the microscopes used historically, they are used for much the same purpose: viewing objects that would otherwise be indiscernible to the human eye. Here we’ll start with a basic compound microscope and go on to explore the components and function of larger more complex microscopes. We’ll also take an in-depth look at one of the key parts of a microscope, the objective lens.
In many microscopes, backlight illumination is favored over traditional direct light illumination due to the latter’s tendency to over-saturate the object under inspection. One specific backlight illumination technique employed in microscopy is Koehler illumination. This method involves flooding the object with light from behind using incident light from a source like a light bulb (see Figure 2). Koehler illumination utilizes two convex lenses, the collector lens and the condenser lens(or called field lens) , to ensure even and bright illumination on both the object and image planes. This design prevents imaging the light bulb filament, a common issue with direct light illumination. Backlight illumination is also commonly referred to as brightfield illumination.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.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.
- Field of View(FOV): FOV refers to the portion of the object captured by a microscope system. The size of the FOV is dictated by the objective magnification. When employing an eyepiece-objective system, the FOV initially observed through the objective is enlarged by the eyepiece for visual examination. In a camera-objective system, this FOV is transmitted onto a rectangular camera sensor. Due to the sensor’s shape, it can only capture a segment of the complete circular FOV from the objective. In contrast, the human eye’s retina can capture a circular area, encompassing the entire FOV. Consequently, the FOV produced by a camera-microscope system tends to be slightly smaller than that of an eyepiece-microscope system.
- 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.