Microscopes in Action

Resolution, contrast and illumination

Human Chronic Myelocytic Leukemia

The Importance of Resolution and Contrast in Microscopy

There are two primary considerations for microscope optics to make very small things visible: resolution and contrast.

Resolution is defined as the ability to visually separate two points. This is complicated by the fact that very small magnified points do not actually appear as points, but as small patterns called Airy patterns, or discs, due to the wave nature of light and its diffraction

The resolution limit of a given microscope objective is based on the numerical aperture of the objective and the wavelength of light used. It can be expressed by a mathematical equation and is more or less fixed for each objective under normal circumstances.

Contrast, on the on the other hand, is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity. Simply put, contrast is the difference between two colors. 

So, two points, even with adequate contrast, can be so small and close together as to appear as one. Fine details of this size will not be seen. As show below, two points, otherwise large enough to be resolved, may not be seen if there is insufficient contrast between them and the background.

In microscopy, resolution is more or less fixed, while contrast can easily be manipulated using various lighting techniques as shown below.

Contrast Effects

This image shows eight rows of letters. Each successive row is a little lighter shade relative to the previous row, and closer in shade to the background white.

The top row is black, and has the highest contrast with the white background.

The bottom row, which on some displays may not show due to the low contrast, is a very light grey, almost indistinguishable from the background white. If you are having a hard time seeing it, it is R S Z H V R. It is visible on my regular desktop PC monitor, but not my laptop screen.

If we were looking at the letters under a microscope, each is clearly big enough to see, so resolution is not an issue. What is an issue is our ability to see the letter due to its low contrast against the white background. It works the same way with colors. Some combinations are high contrast and easy to see. Others look so much alike they seem to blend together as with the very light grey last row here.

As you might imagine, the fine structures of microscopic organisms, or even thin sections of larger organisms, are often nearly transparent, and can show very little contrast against the background illumination, particularly if it is bright. They can easily be as difficult to see, if not more so, than the last row of letters in the accompanying image. The job of the microscopist is to use a method of illumination that provides the necessary contrast to see the organisms and structures of interest.

Making it Easier to See:
Illumination and Contrast Methods in Microscopy

Brightfield: This is the term used for normal microscope illumination. The light source is below the specimen and shines up through it finally resulting in the image in the eyepieces.  Most of the microscope images you have seen are brightfield images. From Wikipedia: https://en.wikipedia.org/wiki/Bright-field_microscopy  And from Olympus, we have an additional explanation of the technique and a brightfield image gallery: https://en.wikipedia.org/wiki/Bright-field_microscopy

Darkfield: Darkfield is the term used when the objects are made to appear bright, on a dark to black background. This is done by shining a cone of light up through the specimen so it is illuminated from the sides. This results in the specimen appearing to glow against the black background. This is accomplished either by using a special darkfield condenser that has the center portion blocked off, or an opaque “patch stop” which is just a circular device placed in the light path to block the center portion of the light column, leaving just a circular ring of light to go up through the condenser and illuminate the specimen. The fact that the condenser refracts the ring of light toward the center converts it to the cone of light necessary to illuminate the specimen from the sides. This method can produce beautiful images, particularly of specimens such as diatoms and is quite inexpensive to implement using the patch stop method. From Olympus: https://www.olympus-lifescience.com/en/discovery/what-is-darkfield-microscopy/

Oblique Contrast: Also called anaxial (asymetrical or off-axis) contrast, oblique contrast is a very old technique that uses only a part of the condenser light cone to illuminate the specimen from a side angle, resulting in a higher contrast, more 3-dimensional appearance. Also an inexpensive technique to employ using a patch stop of some form, the appearance can be changed at will by rotating the patch stop to allow the light to illuminate the specimen from different angles, or by using different patch stops. The resulting image usually does not have the dark background found in darkfield, but shares some of the same techniques. Various forms of patch stops can be utilized, and the technique can have variable effects depending upon the specimen. In a variation called circular oblique illumination the patch stop is made to allow a complete or nearly complete circular cone of light to fall on the specimen. These patch stops resemble phase contrast condenser annuli in that they are complete circles. From Olympus: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/oblique/obliqueintro/ From Molecular Expressions Microscopy Primer: https://micro.magnet.fsu.edu/primer/techniques/oblique/obliqueintro.html

Rheinberg Illumination: This technique is like Darkfield, with the difference being the patch stops are colored. Instead of an opaque central stop and clear surroundings, the central stop is a fairly dark but transparent color, often dark blue. The outer ring is a lighter transparent and contrasting color. Olympus has a nice description here: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/rheinberg/ The McCrone Institute has a nice article on Rheinberg here: https://www.mccrone.com/mm/rheinberg-illumination-for-high-color-contrast/

Polarization Microscopy: Polarization of light is a very complex subject. Polarization microscopy in its most basic form uses two linear polarizers. One is inserted in the light path below the specimen, usually below the condenser, and the other is inserted near the top of the light path above the objective but below the eyepiece. The second polarizer, called the analyzer, can be used above the eyepiece as well. Depending on the particular use, the polarizers may be oriented with the polarization axes parallel, crossed at 90 degrees, or somewhere in between. Its most common use is in petrology, the study of rocks and the processes that form and transform them. It can also be used as a contrast enhancing method, either by itself or in combination with phase, oblique, or darkfield techniques. Basic polarization microscopy is inexpensive, requiring only two pieces of polarizing material. High-end polarization microscopes are very complex and quite expensive. Nikon’s MicroscopyU has a very nice explanation here: https://www.microscopyu.com/techniques/polarized-light/polarized-light-microscopy

Phase Contrast: This is a method developed in the 1930’s, and first published in 1942 by Fritz Zernike who went on to win the 1953 Nobel Prize in Physics, for the discovery. When light passes through a specimen and the media surrounding it, phase shifts will occur in the light depending on what it interacts with. Thicker or more dense parts of the specimen result in phase changes different from those induced by light shining through thinner parts or the media surrounding the specimen. Phase Contrast uses specially constructed objectives and condensers to convert those phase shifts, which are invisible to the eye, to amplitude, or brightness, shifts which the eye can then see. Nikon’s MicroscopyU has a nice description here: https://www.microscopyu.com/techniques/phase-contrast/phase-contrast-microscope-configuration Olympus has a nice section, including an image gallery, here: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/phasecontrast/phaseindex/

Differential Interference Contrast (DIC): Differential Interference Contrast, also called Nomarski Interference Contrast, is a high-end, very expensive, optically complex research technique. It uses polarized light and specially constructed prisms. The light first passes through a linear polarizer, in some applications next in the light path is a wave plate/retarder. Then the light passes through the condenser, the specimen, into the objective, through the second special prism, the analyzer (second polarizer), then to the eyepiece. Wikipedia has a good explanation here: https://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy Olympus has a very nice primer on DIC microscopy here: https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/dic/dicintro/ iBiology has a nice YouTube video by Professor Edward Salmon here: https://www.youtube.com/watch?v=FUa1GTc69y4

Fluorescence Microscopy: This is an advanced research technique that uses fluorescent dyes to label particular cells or parts of cells. Fluorescense utilizes these special dyes to “light up” the areas or cells of interest. The technique generally requires use of an antibody to a particular cell type or part of a cell. The fluorescent dye is attached to the antibody so when the antibody attaches to the target, it attaches the dye as well, thereby very specifically “staining” the target area to make it stand out from the background. Then, by shining a particular, longer wavelength light onto the specimen and viewing image provided by the resulting shorter wavelength light emitted by the dye, you can identify the target area of interest. This technique is also the basis for the newest techniques known as super resolution microscopy. Nikon has a nice introduction to fluorescence microscopy here: https://www.microscopyu.com/techniques/fluorescence/introduction-to-fluorescence-microscopy

Reflected Light (Episcopic) Illumination: Reflected light illumination, often called epi-illumination, is designed for use on non-transparent specimens. Anything from metal surfaces, computer chips, paint, paper, wood, and pretty much anything with a non-transparent surface. The specimen is lighted from above, with the light shining down through the objective, then reflecting off the specimen and back up through the light path to the eyepieces. Illumination from below, as in normal brightfield microscopy is called diascopic illumination. Standard techniques like brightfield, darkfield and DIC can be used in conjunction with reflected light illumination. Normal fluorescense microscopy utilizes reflected light. Nikon’s MicroscopyU has a nice explanation here: https://www.microscopyu.com/techniques/stereomicroscopy/reflected-episcopic-light-illumination Olympus has a description as well: https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/reflected/

Stereo Microscopy: These microscopes, often referred to as dissecting microscopes, have two complete light paths, one for each eye. Some, particularly the older ones, have two complete microscopes side-by-side, at slightly converging angles. The newer ones have two eyepiece and upper tube assemblies, but only one objective which is ground in such a way to provide separate light paths to the eyepieces. The two separate light paths give two separate images, and allow for stereo, or 3D views of the specimen. The effect is like that of binoculars. Standard microscopes only have one light path and produce one image, even if it has a binocular head, and the image in one eyepiece is the same as the image in the other. Stereo or 3D views are not possible. Stereo microscopes have much longer working distances and generally lower maximum magnification. Some even have camera ports for still or video photography, and pictures can always be taken through one eyepiece with your cell phone. I shot this video  of a Damselfly nymph in a petri dish of pond water, using an Olympus SZH stereo microscope: https://www.youtube.com/watch?v=OMNHuab9hKE

Stereo microscopes make great starter microscopes for kids as you can look at most anything you can get under the microscope, and they’re much less difficult to handle. Insects, worms, flowers, leaves, rocks,  sand, money, stamps, pictures, paper, cloth, animal bones, or even hands and fingers make fine objects for curious students. Nikon’s MicroscopyU has a very nice introduction here: https://www.microscopyu.com/techniques/stereomicroscopy/introduction-to-stereomicroscopy