Custom integration projects have specific performance requirements that a commercially available microscope alone cannot provide. Although microscope components are key to the product’s design and performance, selecting these parts can be confusing. Understanding the key building blocks of integrated systems and the common nomenclature used by optics manufacturers will save product developers both time and money during the component selection process. The best way to start is to review the proposed system and its intended output. Any or all of three main types of components – optics, illumination sources and imaging detectors – may be required. Often, standard components are assembled and custom-mounted together (Figure 1). Figure 1. A reflected-light illuminator, objective lens, nosepiece and trinocular viewing tube lens comprise a standard microscope configuration often used with custom mounting. Images courtesy of Olympus America Inc. Optics Not only are performance specifications such as resolution and transmission important, but also mechanical and space considerations are crucial. Whether the system must deliver a specified spatial resolution for measurement or achieve optimal visual contrast for imaging or spectral data, manufacturers use consistent terminologies when describing optics. These include: Infinite vs. finite optical systems Most optical systems use an infinity-corrected optical design to make it easier to add intermediate components. In these systems, the intermediate image plane is formed by the tube lens, so there are parallel light rays between the objective lens and tube lens. In finite systems, the intermediate image planeis formed by the objective (Figure 2). Infinite systems allow prisms, sliders and other components to be introduced without affecting magnification. There is, however, a fixed range for the distance between the objective lens and tube lens, so it is wise to ask the supplier for the parfocal length of the objective lens and the focal length of the tube lens. In addition, knowing the recommended maximum separation between the tube lens and the objective lens allows the system designer to achieve maximum performance without affecting illumination uniformity. Figure 2. Comparison between infinity-corrected (above) and finite-corrected (below) optical systems. Numerical aperture Numerical aperture (NA) describes the inherent ability of an objective lens to gather light and resolve fine detail. The higher the NA, the better the resolving power of the lens.1 Objectives with higher NAs tend to be more expensive. Lateral resolution Resolution of a microscope is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities.2 Resolution also depends on the system’s wavelength as defined by the Rayleigh equation where R is the distance between two adjacent particles, λ is the wavelength of light and NA is the numerical aperture of the objective. Working distance Working distance is the distance between the glass element of an objective’s mounting structure and a focused specimen. It is an important consideration when working with topographical samples that have changes in height, to help avoid any possibility of the objective contacting the sample. Focal length Focal length is important for understanding the total magnification of the system, which is the ratio of the focal length of the tube lens to the focal length of the objective lens. To change magnification, the tube or the objective lens can be changed. In most cases, it is easier to change the objective lens because there typically is more selection from manufacturers, enabling designers to avoid custom design costs; in cases where there is a specific objective lens performance characteristic that must be preserved, the tube lens can be changed. Figure 3. Example systems based on standard optical components for integration into larger pieces of equipment. The system on the left shows a tube lens with eyepieces for operator viewing, and the system on the right includes an autofocus unit and a tube lens with a direct camera mount. Transmission Most objective lenses are designed for peak transmission at a specific wavelength. Outside of this range, performance can drop off significantly, so matching the system wavelength to the appropriate objective is critical to system performance. Many objective lenses are corrected for the visible light spectrum (400 to 700 nm). Other specialized objectives peak in the ultraviolet (700 nm) ranges. Illumination sources Depending upon the system, illumination may come from a laser, fiber optic lightguide, or standard halogen or mercury bulb. There are three key factors in selecting the appropriate light source for a system. First is the wavelength and size of the light source. The wavelength must be complementary to the output. Additionally, the light source must provide sufficient energy to transfer through the system and be returned to the sensor. Second are the optical characteristics of the system, including projection lens magnification and the exit pupil diameter of the objective lens. Each projection lens assembly has a magnification that optimally projects the light to the back focal plane of the objective lens. Current practice for spatial measuring and imaging systems is to use Köhler criteria, to provide even distribution of light from the source across the back focal plane of the lens, but some systems use critical illumination – which relays the illumination source directly to the back focal plane – for optimum efficiency when gathering spectral data. The exit pupil should be entirely filled by the illumination, and pupil diameters vary from objective to objective. Many manufacturers may provide projection lens magnification and exit pupil – back focal plane location – diameters so that the illuminator/projection lens can be matched to the objective lens. Figure 4. Both the optical performance and the mechanical dimensions of objective lenses must be considered in the overall design. The differences in mechanical dimensions of an objective lens series can be seen. Third is the type of illumination. For metrology, fiber optics and standard halogen lamphouses are commonly used. For applications requiring high-intensity light, a metal-halide power supply can be more effective than traditional mercury bulbs. High-intensity light sources, with their longer bulb life and better illumination stability, use coherent fiber guides. Collimating adapter lenses are used to optimize the light path from the fiber guides through the optics of the microscope by focusing the light into parallel rays. Another illumination source gaining in popularity is the LED, which can be automated for simple intensity control and fast on/off switching. LEDs offer high reliability, long lifetimes and low energy usage, making them both practical and better for the environment. An additional advantage of LED illumination is that there is no change in color temperature when adjusting the voltage, so the system maintains continuous color fidelity, unlike traditional halogen sources that appear yellow at low-voltage power levels. From an integrator’s standpoint, there are other factors to consider. Each optical component or detector is rated to withstand a certain amount of energy or heat. When working with high-power illumination sources such as lasers, it is important to identify any limitations of the optical coatings or other structural elements. For temperature-sensitive samples, it is desirable to remove as much heat as possible. Using an external light source such as a fiber guide and a cooled power supply or LED illuminator removes heat from the system, helping to preserve the sample’s integrity. Imaging detectors Not all systems require imaging components, but for many applications, it is standard to view, capture and create an imaging database. If imaging is the primary means for data collection, having the right detector is vital. The following are some parameters to consider when selecting a suitable digital imaging device: Digital resolution Many people are misled into thinking that, when it comes to megapixels, bigger is better; i.e., that having more megapixels means gaining better resolution. Ultimately, designers must match the optical resolution of the microscope system or spot size projected on the CCD with the digital resolution of the camera to avoid over- or under-sampling. The spot size of the projected image is dependent upon the objective lens magnification, numerical aperture, relay lens design and tube lens. The resolution of the CCD depends on both the size and number of pixels. The desired optical resolution should be considered first, and then digital spatial resolution should be determined, as it is the limiting factor in achieving the system’s overall resolution. This phenomenon is explained by Nyquist’s criterion, which says that two to three pixels on a CCD chip are required to resolve the smallest feature that the optical system can reproduce. Using a smaller number of pixels results in image degradation. A larger number of pixels does not assist in resolution and may be more costly. Dynamic range Dynamic range relates to the ability of the sensor to collect data at very low and very bright levels simultaneously. Spectral response Just as it is important to match the wavelength of the emitting light source with the peak performance range of the objective lens, it also is important to match the light source to the detector. An imaging device’s spectral sensitivity is measured by its quantum efficiency, which is the probability that the detector will detect a photon of a given wavelength, expressed graphically. Cooled vs. uncooled Depending upon the application, cameras may require cooling. Options include liquid nitrogen, water, air and Peltier thermoelectric cooling. Some of the choices require additional space and/or external equipment. Cooled cameras are more sensitive at low light levels and usually are more expensive. Integration considerations A digital camera requires software to operate. Computer and interface options include such standards as PCI, USB, FireWire and Ethernet, all of which have advantages and disadvantages. Data collection from the digital camera also may have to be synchronized with external hardware devices such as a mechanical shutter or laser pulse. Understanding the basic building blocks of optical and digital microscopy, the application’s design parameters and how they are interrelated can help developers ensure that integrated systems meet the needs of their users in the most efficient and effective way. Besides technical specifications, it is vital to work with manufacturers with OEM integration experience, as careful dedication and planning are necessary to provide controlled pricing and to meet demanding delivery logistics throughout the product life cycle. Meet the author Jennifer Wrigley is associate product manager at Olympus America Inc. in Center Valley, Pa.; e-mail: jennifer.wrigley@olympus.com. References 1. www.olympusamerica.com/seg_industrial/files/industrial_component_guide.pdf. 2. For more information, consult: www.olympusmicro.com/primer/anatomy/numaperture.html.