Proper Lighting Gets the Most from Microscope Images

But optical components alone don’t limit image quality. How you illuminate a sample makes a tremendous difference in the appearance of images you acquire from a microscope. You want the best image you can get so it will clearly show the features you need to examine.

Two factors play key roles in obtaining good images: the source direction of the light that illuminates your sample, and the contrast medium you use to reveal details of the sample.

Figure 1 shows the terms microscopists generally use to describe the directions of light used to illuminate a sample. When most people think of microscope images, they think of those produced by brightfield illumination that passes up through a sample. Different parts of a semitransparent sample attenuate or color the transmitted light, thus forming an image.

Brightfield illumination allows some of the light from the light source to reach the microscope objective. In a biological microscope, for example, a manufacturer places a light source on the optical axis and shines light toward the microscope objective. Thus, most of the light enters the microscope optical system directly.

People who work with biological samples favor brightfield illumination for samples of moderate optical thickness, a term that defines the fraction of light removed from the beam as it passes through the sample. To provide useful visual information, different parts of a sample must have different optical thicknesses.

Although brightfield illumination from below a sample works well for transparent specimens, in all likelihood, you’ll illuminate samples of electronic components from above. The microscope then gathers light reflected from the sample. Reflections come in two types; specular reflections from flat shiny surfaces, and scattering reflections that come from nonuniform surfaces. When you’re lighting a sample from above, you can choose to shine light on the sample at right angles (side illumination), directly down on an object (on-axis illumination), or from an in-between angle (oblique illumination).

Samples Aren’t Transparent
Many microscopes used to examine electronic components incorporate a light source within the microscope housing. A light source projects light into the microscope optics and a half-silvered mirror reflects the light onto the sample from the top (Fig. 2), providing on-axis illumination from above. Although light does not enter the

Figure 1. Brightfield illumination makes images with transmitted light while darkfield illumination uses reflected light.

Figure 2. A built-in brightfield illumination source reflects light onto a sample using a half-silvered mirror. The illuminating light uses the same path as the light returning to the objective lens.

Figure 3. A semiconductor device viewed using brightfield illumination lacks enough contrast to clearly define surface features, although you can distinguish different types of devices. (Courtesy of Mortimer Abramowitz, Olympus.)

Figure 4. A special mirror in a darkfield illumination microscope directs light to a sample without directing any light to the objective. The light travels its own path to the sample.

Figure 5. When viewed using a darkfield light source, the surface of a semiconductor device clearly shows surface features with different reflectances. (Courtesy of Mortimer Abramowitz, Olympus.)

Figure 6. A ring light will illuminate a sample from 360° to help eliminate most shadows. Position the lamp one lamp radius above the sample for illumination at a 45° angle.

 Microscope manufacturers achieve another form of illumination—darkfield illumination—by blocking light from reaching the objective directly from the light source. The optical arrangement in Figure 4 shows how off-axis light reflects from a mirror onto a sample. Note that a hole in the mirror prevents light from reflecting into the objective. The microscope uses a separate lens to focus light on a sample, so light from the source never passes through the viewing optics. In darkfield microscopy, only light reflected from the sample reaches the objective.

Figure 5 shows a darkfield image of a semiconductor device. The image exhibits high contrast so you can quickly differentiate between surface features that have different amounts of reflectance. When a microscope provides darkfield illumination, only light that has touched the sample, and is therefore useful in making an image, gets through the system. Thus, the microscope lets you make useful images of samples that reflect very little light. Brightfield images of such samples would get “swamped” with light from the source.

You can also obtain oblique darkfield illumination from an external light source. This type of oblique illumination reduces specular reflections from flat surfaces, so it provides more useful visual information about a sample. In general, when you use oblique darkfield lighting, you’ll observe more color in images, and you’ll perceive more depth due to shadows cast by three-dimensional features. Those shadows, however, can hide areas you want to inspect. Remember, you’re not aiming for a pleasing, artistic image. You want information about a sample.

Remove the Shadows
If you need to remove those pesky shadows, consider using a ring light, usually a fluorescent light tube shaped into a circle. The tube illuminates the sample from all angles around its circumference and thus eliminates most shadows. Generally, you center a ring light on the sample and place the lamp one lamp radius above the sample (Fig. 6). That arrangement produced oblique illumination at a 45° angle so the microscope can deliver a bright, shadow-free, glare-free image of a sample.

No matter what type of illumination source you choose, you won’t see anything of interest unless your sample modifies the light that reaches the microscope. I’ve illustrated three “contrast mechanisms” above:

1. Images using brightfield light that passes through a sample show the effect of optical thickness.

2. On-axis darkfield-illuminated images show the effect of surface textures on reflected light.

3. Finally, obliquely lit samples show 3-D surface features by highlighting surfaces and emphasizing shadows.

You can use almost any property of the surface or material that affects the light entering the objective to produce contrast in an image. When thinking about the contrast mechanism to use for your inspection, first ask, “What do I want to learn about the sample?” Then, you can look for a way to illuminate the samples to best show the properties you want to learn about.

Explore Other Contrast Mechanisms
If the information you want from a sample has to do with morphology—the shapes of features on your sample—then experimenting with lighting arrangements will usually reveal the best contrast mechanism. If you need information about chemical makeup or pigmentation, look for lighting that lets colors show up properly.

You don’t need to limit yourself to conventional forms of lighting. You can illuminate a sample with ultraviolet light, for example, and observe fluorescence. Many compounds used in electronic products emit specific wavelengths of visible light when illuminated by ultraviolet light. You can use fluorescence to discriminate between these compounds and any surrounding material that has similar optical density, surface texture, and color.

Other contrast mechanisms can exploit changes in phase of coherent light beams and polarization shifts. And you can electrically excite a sample and look for emission of photons. In effect, the electrical stimuli “light” the sample, and the microscope gathers emitted photons. Areas of high emissions usually reveal high current flows and, thus, faults. (This technique requires specialized microscopes that gather low levels of light.) T&MW

FOOTNOTES
1. Masi, C.G., “Microscopes Rely on Basic Optical Components,” Test & Measurement World, April 2000. pp. 38-46. www.tmworld.com/2000/04_microscopes1.htm.  

2. Masi, C.G., “Basic Optical Effects Limit Image Quality,” Test & Measurement World, May 2000. pp. 73–78. www.tmworld.com/2000/05_microscopes2.htm.   

3. Masi, C.G., “Light Characteristics Limit Optical Quality,” Test & Measurement World, June 2000. pp. 77–82. www.tmworld.com/2000/06_microscopes3.htm.

C.G. Masi works as a freelance technical journalist. He is the former chief editor of Test & Measurement World. E-mail: tmw@cahners.com.