Understanding Microscopes and Objectives
A microscope is an optical device used to image an object onto the human eye or a video device. The earliest microscopes, consisting of two elements, simply produced a larger image of an object under inspection than what the human eye could observe. The design has evolved over the microscope’s history to now incorporate multiple lenses, filters, polarizers, beamsplitters, sensors, illumination sources, and a host of other components. To understand these complex optical devices, consider a microscope’s components, key concepts and specifications, and applications.
Components of Microscopes
A compound microscope is one that contains multiple lens elements. It works similar to a simple magnifier which utilizes a single lens to magnify a small object in order for the human eye to discern its details. With a simple magnifier, the object is placed within the focal length of the single lens. This produces a magnified, virtual image. With a microscope, a relay lens system replaces the single lens; an objective and an eyepiece work in tandem to project the image of the object onto the eye, or a sensor – depending upon the application. There are two parts to a microscope that increase the overall system magnification: the objective and the eyepiece. The objective, located closest to the object, relays a real image of the object to the eyepiece. This part of the microscope is needed to produce the base magnification. The eyepiece, located closest to the eye or sensor, projects and magnifies this real image and yields a virtual image of the object. Eyepieces typically produce an additional 10X magnification, but this can vary from 1X – 30X. Figure 1 illustrates the components of a compound microscope. Additionally, Equation 1 demonstrates how to calculate the overall system magnification. In Equation 1, m is magnification.
Eyepieces
When microscopes were first invented, eyepieces played a major role in their design since they were the only means to actually see the object under inspection. Today, analog or digital cameras are used to project an image of the object onto a monitor or a screen. Microscope eyepieces generally consist of a field lens and an eye lens, though multiple designs exist that each creates a larger field of view (FOV) than a single element design. For a simple guide on selecting the right design, view Choosing the Correct Eyepiece.
Illumination
Illumination within a microscope is just as important as selecting the proper eyepiece or objective. It is crucial to choose the correct illumination in order to obtain the most conclusive results. Before deciding on the type of illumination setup to work with, consider the application setup, object under inspection, and desired results.
Many microscopes utilize backlight illumination compared to traditional direct light illumination because the latter usually over-saturates the object under inspection. A specific type of backlight illumination used in microscopy applications is Koehler illumination. In Koehler illumination, incident light from an illumination source, such as a light bulb, floods the object under inspection with light from behind (Figure 2). It employs two convex lenses: the collector lens and the condenser lens. It is designed to provide bright and even illumination on the object plane and on the image plane where the image produced from the objective is then reimaged through the eyepiece. This is important because it ensures the user is not imaging the filament of the light bulb. Since backlight illumination floods the object with light from behind, it is also referred to as brightfield illumination.
Brightfield illumination requires a change in opacity throughout the object. Without this change, the illumination creates a dark blur around the object. The end result is an image of relative contrast between parts of the object and the light source. In most cases, unless the object is extremely transparent, the resulting image allows the user to see each part of the object with some clarity or resolution. In cases where an object’s transparency makes it difficult to distinguish features using brightfield illumination, darkfield illumination can be used.
With darkfield illumination, direct rays of light are not sent into the objective but instead strike the object at an oblique angle. It is important to keep in mind that these rays still illuminate the object in the object plane. The resultant darkfield illumination image produces high-contrast between the transparent object and the light source. When used in a microscopy setup, darkfield illumination produces a light source that forms an inverted cone of light blocking the central rays of light but still allowing the oblique rays to light the object. Figure 3 illustrates a sample darkfield illumination setup where the hollow cone of light is the numerical aperture of the objective. By comparison, no rays are blocked in a brightfield illumination setup. The design of darkfield illumination forces the light to illuminate the object under inspection, but not to enter the optical system, making it better for a transparent object.
A third type of illumination used in microscopy is epi-Illumination. Epi-illumination produces light above the objective. As a result, the objective and epi-illumination source substitute for a Koehler illumination setup. Using the objective for a large section of the illumination makes epi-illumination very compact – a major benefit of this design. Figure 4 illustrates an epi-illumination setup that is used frequently in fluorescence applications. For more information on fluorescence microscopy, view Fluorophores and Optical Filters for Fluorescence Microscopy.
Objectives
Objectives allow microscopes to provide magnified, real images and are, perhaps, the most complex component in a microscope system because of their multi-element design. Objectives are available with magnifications ranging from 2X – 200X. They are classified into two main categories: the traditional refractive type and reflective. Each category is further divided into types: finite conjugate and infinite conjugate (infinity corrected). In order to choose the correct objective, it is important to know the benefits of one category and type from another.
Objectives: Refractive
The most commonly used category of objectives is refractive. In a refractive design light passing through the system is refracted, or bent, by the optical elements. Each optical element is typically anti-reflection coated to reduce back reflections and improve overall light throughput. Refractive objectives are often used in machine vision applications that require resolution of extremely fine details. There are multiple refractive objective designs each utilizing different optical configurations. The designs can range from two elements in basic achromatic objectives (an achromatic lens and a meniscus lens) to fifteen elements in plan-apochromatic objectives (Figure 5). Plan-apochromatic objectives are the most complex, high-end objective design with chromatic and flat field correction done within the objective itself.
Objectives: Reflective
Reflective objectives utilize a reflective, or mirror-based design. They are often overlooked in comparison to their refractive counterparts, though they can correct for many issues present in the latter. Reflective objectives consist of a primary and secondary mirror system (Figure 6) to magnify and relay the image of the object under inspection. Edmund Optics® utilizes the popular Schwarzschild design, though other designs are available. Since light is reflected by metallic surfaces and not refracted by glass surfaces, reflective objectives do not suffer from the same aberrations as refractive objectives and, thus, do not need the additional designs to compensate for these aberrations. Reflective objectives can produce higher light efficiency as well as better resolving power for fine detail imaging because the system is primarily dependent upon the mirror coating instead of upon the glass substrate being used. Another benefit of reflective objectives is the possibility of working deeper into either the ultra-violet (UV) or infrared (IR) spectral regions due to the use of mirrors compared to conventional refractive optics.
Key Concepts and Specifications
Most microscope objective specifications are listed on the body of the objective itself: the objective design/standard, magnification, numerical aperture, working distance, lens to image distance, and cover slip thickness correction. Figure 7 shows how to read microscope objective specifications. Since the specifications are located directly on the body of the objective, it is easy to know exactly what one has, a very important fact when incorporating multiple objectives into an application. Any remaining specifications, such as focal length, FOV, and design wavelength, can easily be calculated or found in the specifications provided by the vendor or manufacturer.